Mierob Ecol (1990) 19:211-225
MICROBIAL ECOLOGY @Springer-VerlagNew York Inc. 1990
Kinetics of Ammonia Oxidation by a Marine Nitrifying Bacterium: Methane as a Substrate Analogue B. B. Ward Marine Sciences Program, Universityof Californiaat Santa Cruz, Santa Cruz, California95064, USA
Abstract.
In pure culture, the marine ammonia oxidizer, N i t r o s o c o c c u s oceanus, exhibits normal Michaelis Menten kinetics with respect to its primary substrate, ammonia. N. o c e a n u s also exhibits a kinetic response to methane. In the absence of methane, oxidation of ammonia is first order with respect to ammonia concentration under atmospheric oxygen concentrations at seawater pH. In the presence of methane, ammonia oxidation is inhibited, and the amount of inhibition is related to the relative concentrations of methane and ammonia. Using semicontinuous batch cultures as a source of organisms for short-term kinetic experiments, I investigated the relationship between ammonia and methane oxidation in N. o c e a n u s by varying the absolute and relative concentration of both substrates. Methane appeared to act as a substrate analogue, and its effect on ammonia oxidation was modeled as a permutation of competitive inhibition involving a cooperative enzyme system. Methane was oxidized by N. oceanus, even in the absence of measurable ammonia oxidation, but the process was inhibited at increasing methane concentrations. Of the two product pools analyzed, an average of 37% of methane oxidized was detected in particulate (cell) material and the remainder was detected in ~4CO2. The contribution of methane to total carbon assimilation varied with the ratio [CH4]/[NH3] and may be significant under substrate concentrations typical of a dilute aquatic environment.
Introduction Nitrosococcus o c e a n u s is considered a true obligate autotroph (sensu Whitten-
bury and Kelly, ref. 31). It is incapable of growth on organic substrates [32], with very minimal incorporation of carbon from complex organic substrates [ 13] and derives all cellular energy and cell carbon from oxidation of ammonia and the fixation of CO2 via the Calvin Benson cycle. N. o c e a n u s is, however, capable of methane oxidation, producing both labeled cell material and CO2 [12, 28]. Methane and ammonia are oxidized simultaneously, but even small concentrations of methane inhibit ammonia oxidation [12, 28]. The mode of inhibition appears to be competitive at high ammonia concentrations, as has been demonstrated for N i t r o s o m o n a s e u r o p a e a [23]. However, at lower con-
212
B.B. Ward
c e n t r a t i o n s o f a m m o n i a , t h e i n t e r a c t i o n a p p e a r s to i n v o l v e s o m e k i n d o f c o o p e r a t i v i t y , a n d n o n - M i c h a e l i s M e n t e n k i n e t i c s are o b s e r v e d [28]. T h e e x p e r i m e n t s d e s c r i b e d b e l o w were d e s i g n e d to d e t e r m i n e t h e n a t u r e o f the i n t e r a c t i o n b e t w e e n m e t h a n e a n d a m m o n i a o x i d a t i o n i n N . o c e a n u s . T h e r e s u l t s are i n t e r p r e t a b l e i n t e r m s o f a m o d e l i n w h i c h m e t h a n e acts as a n a n a l o g u e o f a m m o n i a . S i g m o i d a l k i n e t i c s are a r e s u l t o f d i f f e r e n t i a l affinity for substrate a n d analogue by different configurations of a m u l t i c o m p o n e n t enz y m e . It is h o p e d t h a t the m o d e l , a l t h o u g h s p e c u l a t i v e , m a y y i e l d i n s i g h t s a n d possibly predictions concerning the b e h a v i o r of a m m o n i a - o x i d i z i n g bacteria i n the sea. A s e c o n d o b j e c t i v e was to d e t e r m i n e the fate o f m e t h a n e c a r b o n (cell v s CO2 p r o d u c t i o n ) a n d to assess the c o n t r i b u t i o n o f m e t h a n e o x i d a t i o n to c e l l u l a r c a r b o n m e t a b o l i s m . T h e r e l i a n c e o f N . o c e a n u s o n CO2 f i x a t i o n b y t h e C a l v i n B e n s o n cycle m i g h t b e a m e l i o r a t e d i f it w e r e a b l e t o a s s i m i l a t e CO2 d i r e c t l y f r o m m e t h a n e , e i t h e r v i a f o r m a t e , as it is d o n e b y t r u e m e t h a n o t r o p h s , o r b y r e c y c l i n g CO2 p r o d u c e d f r o m m e t h a n e o x i d a t i o n . T h e r e is n o e v i d e n c e for the p r e s e n c e i n nitrifiers o f e n z y m e s i n v o l v e d i n m e t h a n e o x i d a t i o n b y m e t h a n o t r o p h s . It a p p e a r s , n e v e r t h e l e s s , t h a t N . o c e a n u s d o e s a s s i m i l a t e s o m e carbon from methan e. T h e d e p e n d e n c e of the competitive interaction on b o t h s u b s t r a t e s a n d the fate o f m e t h a n e - d e r i v e d c a r b o n as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f b o t h s u b s t r a t e s is d e s c r i b e d b e l o w .
Methods N. oceanus was grown in 2.5-liter semibatch culture as previously described [28]. Purity of the
culture was monitored by plating onto rich seawater medium or into liquid aliquots of the same medium [51. Contamination would easily have been detected during microscopic examination of samples. N. oceanus is a large coccus, easily distinguished from common contaminants during routine epifluorescent enumeration of experimental samples (see below). If contamination was discovered, the culture was discarded. Only demonstrably pure cultures were used in the experiments reported here. At approximately weekly intervals, 2 liters of culture were harvested and cells were concentrated by filtration onto sterile 1/~m pore size Nudepore filters. Cells were washed off the filters and resuspended in sterile ammonia-free seawater medium [28]. Replicate 80-ml samples of resuspended cells were aliquoted into several 300-ml flasks equipped with serum stoppers. Cell concentration in experimental flasks was about l0 ~ cells ml -~ (see below). Methane (Union Carbode, 99.99%) was added to the headspace by syringe, and, after equilibrium with the medium, methane concentration in the headspace of each flask was measured by flame ionization gas chromatography as described previously [28]. The coefficient of variation for replicate assays of a single flask averaged 4.8%. Dissolved methane concentrations were computed using the solubility data of Yamamoto et al. [33]. RadiolabeUed methane, z4fU4, of high specific activity was produced biogenically by the method of Daniels and Zeikus [8]. The labelled methane was sealed in Hungate tubes containing 1 ml of 10 N NaOH using solid black rubber stoppers and crimp seals and stored upside down. Before use, [abelled methane was removed from the storage tube by syringe and passed through a syringe containing silver oxide crystals into an evacuated tube. Silver oxide is used to scrub CO from the methane preparation (M. I. Scranton, personal communication). Specific activity was determined for each tube by the method of Zehnder et al. [34]. No change in methane content or specific activity was detected during storage of >- 1 yr. Subsamples of the cleaned methane were added by syringe to experimental flasks, and the volume removed from the tracer tube was replaced by sterile water. Experimental flasks were sampled 4 to 6 times over the course of several hours. Cell number (by acridine orange direct counting) and ammonia concentration [14] were measured in duplicate
Effects of Methane on Ammonium Oxidation Kinetics
213
on aliquots taken at time zero. At subsequent time points, 10-ml aliquots of cell suspension were removed by syringe and added directly to tubes containing 0.1 ml 10 N NaOH. The basic aliquots were filtered through 0.45 #m pore size cellulose acetate filters and rinsed with filtered seawater. Filtrates were placed in 160 ml serum bottles and sealed with a phenethylamine laden filter (100 ~tlfresh phenethylamineon a 25-mm GF/C filter) suspended above the liquid. Concentrated sulfuric acid (0.5 ml) was added by syringe and the filtrate swirled occasionally during the next 24 hours. Both filters, one containing the particulate material and one the captured CO2, were analyzed by liquid scintillation counting. Uninoculated controls were incubated and sampled in parallel (medium with labelled methane but without bacteria). In this protocol, the 1"CO2 capture efficiency was found to be 80% after 24 hours. We have found that although the acidified filtrate contains zero counts above background after 24 hours, we do not capture 100% of labeled ~4CO2 on the phenethylamine filter. This phenomenon was investigated--by adding ~4CO2to water and monitoring its removal from the filtrate and capturing it on a filter following acidification--and found to be reproducible. Counts have been corrected for the experimentally determined efficiency. Less than 1% of label in soluble form (either ~4CH4or ~4COz)is retained on filters after addition of 1 or 10 N NaOH. Nitrite concentration was assayed [20] in 1.0-ml subsamples over the time course. No duplicate time point samples were taken, because previous experiments had shown duplicates to be statistically identical. Instead, linear regressions of data over the time course were used to assess the quality of the data (see below). Similarly, only rarely were individual treatments within experiments replicated, because no significant differences were found between replicated treatments previously (see ref. 28), and the amount of labor involved in processing the radiolabelled samples prohibited excessive numbers of samples. CH4 and production of ~4CO2 ( V ~ ) were Rate of methane incorporation into cell material (V~:n,) computed from linear regressions of label accumulation in each pool. Total methane oxidation (Vcm) was computed from the regression of the sum of label in particulates and in ~4CO2at each time. Only t4CO2 and cell pools were quantified. Release of soluble compounds into the medium was assumed to be neglible based on the findings that 1) acidified filtrates always produced counts the same level as the background after 24 hours, and that 2) 100% of 14C-methanol in a strictly analogous experiment was accounted for in cell material plus t4CO2[28]. Rate of ammonia oxidation (VNn3)was computed from the liner regression of nitrite concentrationover time. Rate data reported here are derived from regressions of a minimum of 3 points (P -< 0.05). Rates were normalized to cell number instead of culture volume or some bulk parameter, for example, protein content. Experiments were performed at cell concentrationstoo low to provide adequate biomass for protein determination and, because the cells were derived from semicontinuousculture, they were assumed to be uniform and to have "average" characteristics.
Results A m m o n i a Oxidation Kinetics. I n t h e a b s e n c e o f m e t h a n e , a m m o n i a o x i d a t i o n p r o c e e d e d i n s i m p l e M i c h a e l i s M e n t e n f a s h i o n a n d was a n a l y z e d b y p l o t t i n g m e t h o d s to y i e l d k i n e t i c p a r a m e t e r s [28] (Figs. 1, 2). T h e H i l l p l o t i n d i c a t e s t h a t i n t h e a b s e n c e o f c o m p e t i n g s u b s t r a t e (Fig. 1A), the r e a c t i o n is e s s e n t i a l l y first o r d e r w i t h r e s p e c t to a m m o n i a c o n c e n t r a t i o n i n t h e p r e s e n c e o f o x y g e n at atmospheric e q u i l i b r i u m concentration. In the absence of me th a n e , both the Hill a n d H o f s t e e p l o t s are l i n e a r . I n t h e p r e s e n c e o f m e t h a n e , p o s i t i v e c o o p e r a t i v i t y is i n d i c a t e d b y the i n c r e a s e i n slope o f t h e H i l l p l o t i n t h e m i d d l e s u b s t r a t e c o n c e n t r a t i o n r a n g e (Fig. 1B) [6] a n d t h e p r e s e n c e o f a m a x i m u m i n the H o f s t e e (or S c a t c h a r d ) p l o t (Fig. 2) [6, 15].
Methane Oxidation Kinetics. T h e rate o f m e t h a n e o x i d a t i o n b y N. oceanus varied with methane concentration, but not in a conventional manner. The
214
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0.2 0.4 0.6 0.8 1.0 log [NHa ] Fig. 1. A. Hill plot for oxidation of NH3 in the absence of CH4. Vm,x = maximum velocity; v = initial velocity. Regression by least squares: Iog[v/(Vm~--V)] = --0.92 + 0.97(log[NH3]). r 2 --- 0.99. B. Hill plot for oxidation of NH3 in the presence of 3.5 tzM CH4. Regression by least squares using only the central 3 points, log[v/(Vm.~-v)] = -2.25 + 2.85(log[NH3]). r 2 = 0.98. log [NH a]
t20 100 A
Fig. 2. Hofstee (Scatchard) plot for oxidation of NH3. In the absence (open symbols) of CH4, a straight line results: Vo = 2.01 x l0 -s - 7.58(Vo/[S]), r 2 = 0.98. The equation yields Vm=x(2.01 X 10-8 #M cell-' h -~) and K,, (7.58 ~tM) for this data set directly. In the presence (closed symbols) of 3.5 uM CH4, the relationship exhibits a maximum, rather than linearity.
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d o u b l e r e c i p r o c a l p l o t s f o r d e p e n d e n c e o f V c n 4 o n [CH4] a r e n o t c o n s i s t e n t w i t h M i c h a e l i s - M e n t e n k i n e t i c s (Fig. 3) a n d c a n n o t b e i n t e r p r e t e d to y i e l d k i n e t i c c o n s t a n t s . VcH 4 i n c r e a s e d w i t h i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n but, a t h i g h e r m e t h a n e c o n c e n t r a t i o n s , VcH, d e c r e a s e d . I n h i b i t i o n w a s o b s e r v e d a t d i f f e r e n t m e t h a n e c o n c e n t r a t i o n s in d i f f e r e n t e x p e r i m e n t s , at l e a s t p a r t i a l l y d e p e n d e n t o n t h e a m m o n i a c o n c e n t r a t i o n (Fig. 4). D a t a c o m b i n e d f r o m e x p e r i m e n t s at different ammonia concentrations indicate that methane oxidation rate may b e r e l a t e d t o t h e r a t i o [CH4]/[NH3] (Fig. 5), r a t h e r t h a n t o t h e a b s o l u t e c o n c e n t r a t i o n o f e i t h e r s u b s t r a t e . A t r a t i o s a b o v e a b o u t 12, i n h i b i t i o n is d e t e c t a b l e . However, the relationship between substrate ratio and reaction velocity ratio d e t e r i o r a t e s a t l o w v a l u e s for b o t h r a t i o s a n d is i r r e l e v a n t in t h e c a s e o f CH4 o x i d a t i o n in t h e a b s e n c e o f NH3. M e t h a n e o x i d a t i o n o c c u r s e v e n in t h e a b s e n c e o f N H 3 w h e n n o N H 3 w a s d e t e c t a b l e in t h e m e d i u m a n d n o n i t r i t e p r o d u c t i o n w a s d e t e c t e d o v e r t h e t i m e c o u r s e (Figs. 3, 6) [ 12].
Effects of Methane on Ammonium Oxidation Kinetics
215
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Double reciprocal plot for oxidation
o f CH 4. Lines serve to guide the eye to con-
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Fig. 4. Variation in Vca4 with increasing [CH4] for 3 different experiments, each performed at different [NH3]. [] = 0 ttM NH3; O = 0.24 aM NH3; z~ = 11 aM NH3.
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Fig. 5. Ratio of Vcr~4/VNn3as a function of [CH4]/[NH3]. Data from 3 experiments combined for regression, excluding point (filled circle) at which inhibition by CH4 was evident. N = 14. VcHJVNH3 = 3.998 • 10-4 + 0.004([CHj/INH3]); r 2 = 0.99. Inset shows magnification of values near the origin.
20
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Fate of Methane Carbon. N. oceanus c o n s i s t e n t l y p r o d u c e d
m o r e 14CO2 t h a n CH4 CH4 d i d n o t a p p e a r to b e r e l a t e d to l a b e l l e d cell m a t e r i a l , a n d the r a t i o VcezJVco2 e i t h e r m e t h a n e o r a m m o n i a c o n c e n t r a t i o n a l o n e . T h e r a t i o [CH4]/[NH3] was n o t v e r y i n f o r m a t i v e either: V~c~s cH, as a p e r c e n t o f V c m was v a r i a b l e , b u t h i g h e s t
216
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cm d e m o n s t r a t e s m e t h a n e o x i d a t i o n i n s e ve ra l Fig. 6. A. L i n e a r increase in label ( Vc.4 ~ , plus Vco:) flasks with different [CH4]. S y m b o l s a n d least squares fit to d a t a (Y = CH4 o x i d i z e d as # m o l cell -], X=hours): 9 Y = - 5 . 4 3 x 10 -4 + 5.47 x 10 - 4 X ( r 2 = 0 . 8 8 5 ) ; O = 1.21#M CH4, Y = - 2 . 5 1 x 10 -3 + 4.67 x 10 -3 X (r 2 = 0.989); A = 2.24 # M CH4, Y = - 2 . 9 6 x 10 -3 + 4.87 x 10 -3 X (r 2 = 0.995); O = 3.88 # M CH4, Y = - 3 . 0 3 x 10 -3 + 4.17 x 10 -3 X (r 2 = 0.991); 9 = 4.43 # M CH4, Y = - 1.43 x 10 3 + 3.56 x 10 -3 X (r 2 = 0.996). B. L a c k o f n i t r i t e a c c u m u l a t i o n d e m o n s t r a t e s absen ce o f a m m o n i a o x i d a t i o n , e v e n while m e t h a n e o x i d a t i o n was o b s e r v e d in the s a m e flasks (Fig. 6A). S y m b o l s as for Fig. 6A; a d d i t i o n a l s y m b o l s : 9 = 0 CH4; + = u n i n o c u l a t e d control.
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at highest [CH4]/[NH3] until inhibition in the methane oxidation rate occurred (Fig. 7). Combining all data (including 5 points from the zero [NH3] experiment, which were not included in Fig. 7), an average of 37% of oxidized methane appeared in cells (SD 6.7%,,,N = 20) and 63% in CO2. Other potential pools, such as dissolved organic compounds excreted into the medium or transient internal pools, were not assayed.
RelativeAffinityfor CompetingSubstrates. The slope of the relationship between the ratio Vcm/VNH3 and the ratio [CH4]/[NH3] is the k c J K , , parameter used by Hyman and Wood [11] to compare the affinity ofN. europaea for ammonia and methane. For a single substrate/inhibitor ratio, this parameter was previously found to be 4.3 (in terms of NH3) [28]. Combining all the present data
Effects of Methane on Ammonium Oxidation Kinetics
217
for [CH4]/[NH3] less than 12, the slope is 0.004. This value is much lower than the previous result, which was derived from a single experiment at much lower methane concentration [28]. The present result, the average of several experiments, is the same value that was determined for the kcat/Km parameter for Nitrosomonas europaea [ 11 ]. Although the combined data yield a reasonable correlation coefficient (Fig. 5), significantly different slopes are obtained when each experiment is analyzed separately (not shown). Thus the ratio of substrate Concentrations is not necessarily an adequate empirical basis for prediction of relative reaction rates. The k c a t / K m parameter apparently is not constant and is of limited application in interpreting this interaction.
A Model for A m m o n i a Oxidation in the Presence o f Methane. The pattern of inhibition of ammonia oxidation in N. oceanus by methane may imply the presence of multiple active sites in the enzyme molecule. Very little is known of the geometry of the enzyme, ammonia monooxygenase, because it has proven extremely difficult to purify [18, 24-26]; little progress has been made since the earlier attempts of Watson et al. [30]. The behavior of whole cells may be more applicable than that of purified enzymes to predicting behavior in the environment, even if the underlying nature of the enzyme remains unknown. Adaptations of models that were originally developed to describe purified enZymes have been useful in other kinetic studies in marine systems, notably in nutrient uptake by phytoplankton [2]. The behavior of the whole cell system Under the conditions described here is consistent with the model of active site directed effectors of allosteric enzymes proposed by Smith et al. [19]. In this application, no distinction is made between activity observed in the whole cell and the activity that might be attributed solely to the enzyme (we cannot decipher the possible contribution of, for example, transport systems or feed back control mechanisms involving subsequent enzymes in the oxidative chain). In the model of Smith et al. [ 19], the inhibiting compound (ligand, L) is an analogue of the true substrate (S), and competition between L and S results in sigmoidal kinetics with respect to S. The model describes a cooperative enzyme that occurs in at least two active configurations (El and Ez), each with N active sites (N >- 2). El is the most abundant form of the enzyme and E2 is the least abundant. S binds preferentially to El (K~s -< Kzs) while L binds preferentially to E2 (K2L < K~L). Symbols and definitions are listed in Table 1. In order to compute the predicted VNH3 under conditions of substrate competition, several parameters must be measured or estimated. Some kinetic parameters for ammonia oxidation were measured directly, but others were estimated from the relationships implied by the model. The measured K,~ for ammonia (8 tzM) [28] was used as the Kts of the model, and Kzs (800 ~M) was chosen to reflect the higher affinity of the most abundant form of the enzyme for the primary substrate. Vmaxfor ammonia oxidation also had been measured directly (Table 1) [28]. The value used here is the one obtained from the experiments plotted in Fig. 8 (2.5 x 10 -14 M ) rather than the average value reported earlier (2.2 x 10 -14 M). IS] and [L] used in the model were the actual Substrate and ligand concentrations from the same experiments. In an earlier paper [28], a half saturation constant for inhibition (Ka.) was estimated from the change in Km for ammonia caused by inhibition by methane.
218
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Table 1.
Terms and equations for substrate analogue model [19]
Symbol
Definition
Value calculated 2.5 x 10 -.4 variable variable 1 2 8
E, E~
initial velocity V .... m a x i m u m velocity (M cell -j h -t) substrate concentration ligand concentration monomeric (m = i) or polymeric (m -> 2) n u m b e r of enzyme configurations number of active sites more abundant form of enzyme less abundant form of enzyme
X
[E2]
0.01
go
Vm
Is] [L] m h n
[E,] Dissociation constants
Kts K2s KIL K2L
8 8 18 18
x x X X
10 -6 M 10 -4 M 10 -6 M 10 -7 M
Michaelis Menten equation for reaction velocity in the presence of a competitive inhibitor:
Vm[S]
Vo
K~s(1 +~[L] + [S]) Equation for reaction velocity in presence o f a ligand which is a substrate analog
117]:
vmFts](l
IS]
ELI)~
LK~,~\ +E-d+K-~,d
ts] (1
+xK-~
]Sl + tL] ~
+K~---~ K~,/ J
Vo = 1 +~--dls+K,e]
+X
1 +~s2s+KaL]
Relationships between variables for a ligand which is a substrate analog: K~s < 1; KIL > 1; X < 1
-
This was done to facilitate comparison with work by others in which the relationship between ammonia and methane oxidation had been interpreted as simple competitive inhibition. The K~ so estimated was 6.6 -+ 5.6 uM (N = 6). The uncertainty in this result arises from the necessity to subjectively select data from inhibition experiments which fall on the linear portion of the double reciprocal plots. As can be seen from Fig. 3, this is probably not appropriate, and the Ki so calculated is biased, if not meaningless. Because of conflicting results about the affinity of the enzyme for methane, and the inability to measure affinity constants for the competing substrate directly, it was necessary to estimate the value of K~L based on its relationship to Kls. K1L was estimated to be on the same order as, but slightly higher than K~s, indicating that the affinity of El for the competing substrate (L) is slightly less than its affinity for S. K2L was smaller than K~L, reflecting higher affinity of the least common form of the enzyme (E2) for the substrate analogue (L).
Effects of Methane on Ammonium Oxidation Kinetics
219 B
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2.5
[NH3] 10"a M
Fig. 8, A. Comparison of experimental data (0 and O) and model results (A) for oxidation of NH3 in the presence of 3.5 #M CH4. Model i-esults were generated using the parameters given in Table 2 with [S] in the micromolar range and [L] = 3.5 #M. B. Model results generated using the Parameters given in Table 2, [S] in micromolar range, with varying [L]. O = 7 #M; [] = 4 #M; 9 3.5 uM; A = 2 #M; 9 = 0.1 #M. C. Model results generated using the parameters given in Table 2, except that [S] and ILl are at nanomolar levels, Symbols as in Fig. 8B. D. Model results generated Using parameters given in Table 2, except that affinity constants (Kls, K:s, KiL, K2L) have been reduced by 1,000-fold. [S] and [L] at nanomolar levels. Variable [L]: O = 100 nM; [] = 50 nM; 9 ~20nM;A= 10nM; 9
T h e k e y to p r o d u c i n g s i g m o i d a l k i n e t i c s is m u l t i p l e a c t i v e sites o n t h e e n z y m e m o l e c u l e . B e c a u s e r e l a t i v e l y l i t t l e is k n o w n a b o u t t h i s e n z y m e , t h e m o d e l w a s first r u n b y m a k i n g t h e s i m p l e s t a s s u m p t i o n s a b o u t t h e s y s t e m : a m o n o m e r i c e n z y m e ( m = 1), 2 e n z y m e c o n f i g u r a t i o n s (h = 2), e a c h w i t h 2 a c t i v e sites (n -~ 2). W h e n t h e m o d e l w a s r u n u s i n g IS] a n d [L] p r e v a i l i n g u n d e r e x p e r i m e n t a l COnditions, p r e d i c t e d V v s S p l o t s n e v e r e x h i b i t e d a s m u c h c u r v a t u r e i n t h e low [S] r e g i o n as d i d t h e a c t u a l e x p e r i m e n t a l d a t a . I n Fig. 8 A , it c a n b e s e e n t h a t r e a s o n a b l e fit o f t h e m o d e l t o t h e d a t a is o b t a i n e d w h e n n = 8. W i t h n = 8, t h e m o d e l r e a c t s to i n c r e a s i n g [L] b y i n c r e a s i n g s i g m o i d i c i t y (Fig. 8B), a n d at v e r y l o w [L], e s s e n t i a l l y n o r m a l h y p e r b o l i c k i n e t i c s r e s u l t . A p e r m u t a t i o n o f t h e m o d e l w i t h n = 4 f o r a p o l y m e r i c e n z y m e ( m >-- 2) d o e s n o t p r o d u c e sigmoidicity under the experimental conditions. Thus the configuration necessary to describe the experimental results requires that one monomeric enzyme POSsess s e v e r a l a c t i v e sites. Kinetic parameters which were derived from culture experiments at micro-
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molar levels of [S] and [L], and which adequately explain the kinetic behavior of the system under these conditions, might not be appropriate to the behavior of the organisms under environmental conditions where IS] and [L] are likely to be in the nanomolar range. When the model is run using the kinetic parameters determined in culture but with [S] and [L] in the nanomolar range, no inhibitive effect of methane is seen and no curvature is evident in the V vs S plot of NH 3 oxidation (Fig. 8C). Linear kinetics result because the affinity constants derived from culture are about 1,000-fold higher than environmental IS] and [L]. Obviously, organisms with such low affinities would not be well adapted to exploit an environment with very low substrate concentrations. One possibility to reconcile field and laboratory observations is that organisms in culture are adapted to the high substrate concentrations routinely provided. The same organism might be able to produce enzymes with higher affinities when adapted to growing in a m o r e dilute environment. Environmental substrate concentrations in the range of
MICROBIAL ECOLOGY @Springer-VerlagNew York Inc. 1990
Kinetics of Ammonia Oxidation by a Marine Nitrifying Bacterium: Methane as a Substrate Analogue B. B. Ward Marine Sciences Program, Universityof Californiaat Santa Cruz, Santa Cruz, California95064, USA
Abstract.
In pure culture, the marine ammonia oxidizer, N i t r o s o c o c c u s oceanus, exhibits normal Michaelis Menten kinetics with respect to its primary substrate, ammonia. N. o c e a n u s also exhibits a kinetic response to methane. In the absence of methane, oxidation of ammonia is first order with respect to ammonia concentration under atmospheric oxygen concentrations at seawater pH. In the presence of methane, ammonia oxidation is inhibited, and the amount of inhibition is related to the relative concentrations of methane and ammonia. Using semicontinuous batch cultures as a source of organisms for short-term kinetic experiments, I investigated the relationship between ammonia and methane oxidation in N. o c e a n u s by varying the absolute and relative concentration of both substrates. Methane appeared to act as a substrate analogue, and its effect on ammonia oxidation was modeled as a permutation of competitive inhibition involving a cooperative enzyme system. Methane was oxidized by N. oceanus, even in the absence of measurable ammonia oxidation, but the process was inhibited at increasing methane concentrations. Of the two product pools analyzed, an average of 37% of methane oxidized was detected in particulate (cell) material and the remainder was detected in ~4CO2. The contribution of methane to total carbon assimilation varied with the ratio [CH4]/[NH3] and may be significant under substrate concentrations typical of a dilute aquatic environment.
Introduction Nitrosococcus o c e a n u s is considered a true obligate autotroph (sensu Whitten-
bury and Kelly, ref. 31). It is incapable of growth on organic substrates [32], with very minimal incorporation of carbon from complex organic substrates [ 13] and derives all cellular energy and cell carbon from oxidation of ammonia and the fixation of CO2 via the Calvin Benson cycle. N. o c e a n u s is, however, capable of methane oxidation, producing both labeled cell material and CO2 [12, 28]. Methane and ammonia are oxidized simultaneously, but even small concentrations of methane inhibit ammonia oxidation [12, 28]. The mode of inhibition appears to be competitive at high ammonia concentrations, as has been demonstrated for N i t r o s o m o n a s e u r o p a e a [23]. However, at lower con-
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c e n t r a t i o n s o f a m m o n i a , t h e i n t e r a c t i o n a p p e a r s to i n v o l v e s o m e k i n d o f c o o p e r a t i v i t y , a n d n o n - M i c h a e l i s M e n t e n k i n e t i c s are o b s e r v e d [28]. T h e e x p e r i m e n t s d e s c r i b e d b e l o w were d e s i g n e d to d e t e r m i n e t h e n a t u r e o f the i n t e r a c t i o n b e t w e e n m e t h a n e a n d a m m o n i a o x i d a t i o n i n N . o c e a n u s . T h e r e s u l t s are i n t e r p r e t a b l e i n t e r m s o f a m o d e l i n w h i c h m e t h a n e acts as a n a n a l o g u e o f a m m o n i a . S i g m o i d a l k i n e t i c s are a r e s u l t o f d i f f e r e n t i a l affinity for substrate a n d analogue by different configurations of a m u l t i c o m p o n e n t enz y m e . It is h o p e d t h a t the m o d e l , a l t h o u g h s p e c u l a t i v e , m a y y i e l d i n s i g h t s a n d possibly predictions concerning the b e h a v i o r of a m m o n i a - o x i d i z i n g bacteria i n the sea. A s e c o n d o b j e c t i v e was to d e t e r m i n e the fate o f m e t h a n e c a r b o n (cell v s CO2 p r o d u c t i o n ) a n d to assess the c o n t r i b u t i o n o f m e t h a n e o x i d a t i o n to c e l l u l a r c a r b o n m e t a b o l i s m . T h e r e l i a n c e o f N . o c e a n u s o n CO2 f i x a t i o n b y t h e C a l v i n B e n s o n cycle m i g h t b e a m e l i o r a t e d i f it w e r e a b l e t o a s s i m i l a t e CO2 d i r e c t l y f r o m m e t h a n e , e i t h e r v i a f o r m a t e , as it is d o n e b y t r u e m e t h a n o t r o p h s , o r b y r e c y c l i n g CO2 p r o d u c e d f r o m m e t h a n e o x i d a t i o n . T h e r e is n o e v i d e n c e for the p r e s e n c e i n nitrifiers o f e n z y m e s i n v o l v e d i n m e t h a n e o x i d a t i o n b y m e t h a n o t r o p h s . It a p p e a r s , n e v e r t h e l e s s , t h a t N . o c e a n u s d o e s a s s i m i l a t e s o m e carbon from methan e. T h e d e p e n d e n c e of the competitive interaction on b o t h s u b s t r a t e s a n d the fate o f m e t h a n e - d e r i v e d c a r b o n as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f b o t h s u b s t r a t e s is d e s c r i b e d b e l o w .
Methods N. oceanus was grown in 2.5-liter semibatch culture as previously described [28]. Purity of the
culture was monitored by plating onto rich seawater medium or into liquid aliquots of the same medium [51. Contamination would easily have been detected during microscopic examination of samples. N. oceanus is a large coccus, easily distinguished from common contaminants during routine epifluorescent enumeration of experimental samples (see below). If contamination was discovered, the culture was discarded. Only demonstrably pure cultures were used in the experiments reported here. At approximately weekly intervals, 2 liters of culture were harvested and cells were concentrated by filtration onto sterile 1/~m pore size Nudepore filters. Cells were washed off the filters and resuspended in sterile ammonia-free seawater medium [28]. Replicate 80-ml samples of resuspended cells were aliquoted into several 300-ml flasks equipped with serum stoppers. Cell concentration in experimental flasks was about l0 ~ cells ml -~ (see below). Methane (Union Carbode, 99.99%) was added to the headspace by syringe, and, after equilibrium with the medium, methane concentration in the headspace of each flask was measured by flame ionization gas chromatography as described previously [28]. The coefficient of variation for replicate assays of a single flask averaged 4.8%. Dissolved methane concentrations were computed using the solubility data of Yamamoto et al. [33]. RadiolabeUed methane, z4fU4, of high specific activity was produced biogenically by the method of Daniels and Zeikus [8]. The labelled methane was sealed in Hungate tubes containing 1 ml of 10 N NaOH using solid black rubber stoppers and crimp seals and stored upside down. Before use, [abelled methane was removed from the storage tube by syringe and passed through a syringe containing silver oxide crystals into an evacuated tube. Silver oxide is used to scrub CO from the methane preparation (M. I. Scranton, personal communication). Specific activity was determined for each tube by the method of Zehnder et al. [34]. No change in methane content or specific activity was detected during storage of >- 1 yr. Subsamples of the cleaned methane were added by syringe to experimental flasks, and the volume removed from the tracer tube was replaced by sterile water. Experimental flasks were sampled 4 to 6 times over the course of several hours. Cell number (by acridine orange direct counting) and ammonia concentration [14] were measured in duplicate
Effects of Methane on Ammonium Oxidation Kinetics
213
on aliquots taken at time zero. At subsequent time points, 10-ml aliquots of cell suspension were removed by syringe and added directly to tubes containing 0.1 ml 10 N NaOH. The basic aliquots were filtered through 0.45 #m pore size cellulose acetate filters and rinsed with filtered seawater. Filtrates were placed in 160 ml serum bottles and sealed with a phenethylamine laden filter (100 ~tlfresh phenethylamineon a 25-mm GF/C filter) suspended above the liquid. Concentrated sulfuric acid (0.5 ml) was added by syringe and the filtrate swirled occasionally during the next 24 hours. Both filters, one containing the particulate material and one the captured CO2, were analyzed by liquid scintillation counting. Uninoculated controls were incubated and sampled in parallel (medium with labelled methane but without bacteria). In this protocol, the 1"CO2 capture efficiency was found to be 80% after 24 hours. We have found that although the acidified filtrate contains zero counts above background after 24 hours, we do not capture 100% of labeled ~4CO2 on the phenethylamine filter. This phenomenon was investigated--by adding ~4CO2to water and monitoring its removal from the filtrate and capturing it on a filter following acidification--and found to be reproducible. Counts have been corrected for the experimentally determined efficiency. Less than 1% of label in soluble form (either ~4CH4or ~4COz)is retained on filters after addition of 1 or 10 N NaOH. Nitrite concentration was assayed [20] in 1.0-ml subsamples over the time course. No duplicate time point samples were taken, because previous experiments had shown duplicates to be statistically identical. Instead, linear regressions of data over the time course were used to assess the quality of the data (see below). Similarly, only rarely were individual treatments within experiments replicated, because no significant differences were found between replicated treatments previously (see ref. 28), and the amount of labor involved in processing the radiolabelled samples prohibited excessive numbers of samples. CH4 and production of ~4CO2 ( V ~ ) were Rate of methane incorporation into cell material (V~:n,) computed from linear regressions of label accumulation in each pool. Total methane oxidation (Vcm) was computed from the regression of the sum of label in particulates and in ~4CO2at each time. Only t4CO2 and cell pools were quantified. Release of soluble compounds into the medium was assumed to be neglible based on the findings that 1) acidified filtrates always produced counts the same level as the background after 24 hours, and that 2) 100% of 14C-methanol in a strictly analogous experiment was accounted for in cell material plus t4CO2[28]. Rate of ammonia oxidation (VNn3)was computed from the liner regression of nitrite concentrationover time. Rate data reported here are derived from regressions of a minimum of 3 points (P -< 0.05). Rates were normalized to cell number instead of culture volume or some bulk parameter, for example, protein content. Experiments were performed at cell concentrationstoo low to provide adequate biomass for protein determination and, because the cells were derived from semicontinuousculture, they were assumed to be uniform and to have "average" characteristics.
Results A m m o n i a Oxidation Kinetics. I n t h e a b s e n c e o f m e t h a n e , a m m o n i a o x i d a t i o n p r o c e e d e d i n s i m p l e M i c h a e l i s M e n t e n f a s h i o n a n d was a n a l y z e d b y p l o t t i n g m e t h o d s to y i e l d k i n e t i c p a r a m e t e r s [28] (Figs. 1, 2). T h e H i l l p l o t i n d i c a t e s t h a t i n t h e a b s e n c e o f c o m p e t i n g s u b s t r a t e (Fig. 1A), the r e a c t i o n is e s s e n t i a l l y first o r d e r w i t h r e s p e c t to a m m o n i a c o n c e n t r a t i o n i n t h e p r e s e n c e o f o x y g e n at atmospheric e q u i l i b r i u m concentration. In the absence of me th a n e , both the Hill a n d H o f s t e e p l o t s are l i n e a r . I n t h e p r e s e n c e o f m e t h a n e , p o s i t i v e c o o p e r a t i v i t y is i n d i c a t e d b y the i n c r e a s e i n slope o f t h e H i l l p l o t i n t h e m i d d l e s u b s t r a t e c o n c e n t r a t i o n r a n g e (Fig. 1B) [6] a n d t h e p r e s e n c e o f a m a x i m u m i n the H o f s t e e (or S c a t c h a r d ) p l o t (Fig. 2) [6, 15].
Methane Oxidation Kinetics. T h e rate o f m e t h a n e o x i d a t i o n b y N. oceanus varied with methane concentration, but not in a conventional manner. The
214
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0.2 0.4 0.6 0.8 1.0 log [NHa ] Fig. 1. A. Hill plot for oxidation of NH3 in the absence of CH4. Vm,x = maximum velocity; v = initial velocity. Regression by least squares: Iog[v/(Vm~--V)] = --0.92 + 0.97(log[NH3]). r 2 --- 0.99. B. Hill plot for oxidation of NH3 in the presence of 3.5 tzM CH4. Regression by least squares using only the central 3 points, log[v/(Vm.~-v)] = -2.25 + 2.85(log[NH3]). r 2 = 0.98. log [NH a]
t20 100 A
Fig. 2. Hofstee (Scatchard) plot for oxidation of NH3. In the absence (open symbols) of CH4, a straight line results: Vo = 2.01 x l0 -s - 7.58(Vo/[S]), r 2 = 0.98. The equation yields Vm=x(2.01 X 10-8 #M cell-' h -~) and K,, (7.58 ~tM) for this data set directly. In the presence (closed symbols) of 3.5 uM CH4, the relationship exhibits a maximum, rather than linearity.
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d o u b l e r e c i p r o c a l p l o t s f o r d e p e n d e n c e o f V c n 4 o n [CH4] a r e n o t c o n s i s t e n t w i t h M i c h a e l i s - M e n t e n k i n e t i c s (Fig. 3) a n d c a n n o t b e i n t e r p r e t e d to y i e l d k i n e t i c c o n s t a n t s . VcH 4 i n c r e a s e d w i t h i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n but, a t h i g h e r m e t h a n e c o n c e n t r a t i o n s , VcH, d e c r e a s e d . I n h i b i t i o n w a s o b s e r v e d a t d i f f e r e n t m e t h a n e c o n c e n t r a t i o n s in d i f f e r e n t e x p e r i m e n t s , at l e a s t p a r t i a l l y d e p e n d e n t o n t h e a m m o n i a c o n c e n t r a t i o n (Fig. 4). D a t a c o m b i n e d f r o m e x p e r i m e n t s at different ammonia concentrations indicate that methane oxidation rate may b e r e l a t e d t o t h e r a t i o [CH4]/[NH3] (Fig. 5), r a t h e r t h a n t o t h e a b s o l u t e c o n c e n t r a t i o n o f e i t h e r s u b s t r a t e . A t r a t i o s a b o v e a b o u t 12, i n h i b i t i o n is d e t e c t a b l e . However, the relationship between substrate ratio and reaction velocity ratio d e t e r i o r a t e s a t l o w v a l u e s for b o t h r a t i o s a n d is i r r e l e v a n t in t h e c a s e o f CH4 o x i d a t i o n in t h e a b s e n c e o f NH3. M e t h a n e o x i d a t i o n o c c u r s e v e n in t h e a b s e n c e o f N H 3 w h e n n o N H 3 w a s d e t e c t a b l e in t h e m e d i u m a n d n o n i t r i t e p r o d u c t i o n w a s d e t e c t e d o v e r t h e t i m e c o u r s e (Figs. 3, 6) [ 12].
Effects of Methane on Ammonium Oxidation Kinetics
215
6
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Double reciprocal plot for oxidation
o f CH 4. Lines serve to guide the eye to con-
0 0.0
nect data from individual experiments, but are not meant to imply functional meaning, since the data obviously do not conform to Lineweaver-Burk interpretation.
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Fig. 4. Variation in Vca4 with increasing [CH4] for 3 different experiments, each performed at different [NH3]. [] = 0 ttM NH3; O = 0.24 aM NH3; z~ = 11 aM NH3.
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Fig. 5. Ratio of Vcr~4/VNn3as a function of [CH4]/[NH3]. Data from 3 experiments combined for regression, excluding point (filled circle) at which inhibition by CH4 was evident. N = 14. VcHJVNH3 = 3.998 • 10-4 + 0.004([CHj/INH3]); r 2 = 0.99. Inset shows magnification of values near the origin.
20
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Fate of Methane Carbon. N. oceanus c o n s i s t e n t l y p r o d u c e d
m o r e 14CO2 t h a n CH4 CH4 d i d n o t a p p e a r to b e r e l a t e d to l a b e l l e d cell m a t e r i a l , a n d the r a t i o VcezJVco2 e i t h e r m e t h a n e o r a m m o n i a c o n c e n t r a t i o n a l o n e . T h e r a t i o [CH4]/[NH3] was n o t v e r y i n f o r m a t i v e either: V~c~s cH, as a p e r c e n t o f V c m was v a r i a b l e , b u t h i g h e s t
216
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cm d e m o n s t r a t e s m e t h a n e o x i d a t i o n i n s e ve ra l Fig. 6. A. L i n e a r increase in label ( Vc.4 ~ , plus Vco:) flasks with different [CH4]. S y m b o l s a n d least squares fit to d a t a (Y = CH4 o x i d i z e d as # m o l cell -], X=hours): 9 Y = - 5 . 4 3 x 10 -4 + 5.47 x 10 - 4 X ( r 2 = 0 . 8 8 5 ) ; O = 1.21#M CH4, Y = - 2 . 5 1 x 10 -3 + 4.67 x 10 -3 X (r 2 = 0.989); A = 2.24 # M CH4, Y = - 2 . 9 6 x 10 -3 + 4.87 x 10 -3 X (r 2 = 0.995); O = 3.88 # M CH4, Y = - 3 . 0 3 x 10 -3 + 4.17 x 10 -3 X (r 2 = 0.991); 9 = 4.43 # M CH4, Y = - 1.43 x 10 3 + 3.56 x 10 -3 X (r 2 = 0.996). B. L a c k o f n i t r i t e a c c u m u l a t i o n d e m o n s t r a t e s absen ce o f a m m o n i a o x i d a t i o n , e v e n while m e t h a n e o x i d a t i o n was o b s e r v e d in the s a m e flasks (Fig. 6A). S y m b o l s as for Fig. 6A; a d d i t i o n a l s y m b o l s : 9 = 0 CH4; + = u n i n o c u l a t e d control.
0.5
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0.1 0.0
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at highest [CH4]/[NH3] until inhibition in the methane oxidation rate occurred (Fig. 7). Combining all data (including 5 points from the zero [NH3] experiment, which were not included in Fig. 7), an average of 37% of oxidized methane appeared in cells (SD 6.7%,,,N = 20) and 63% in CO2. Other potential pools, such as dissolved organic compounds excreted into the medium or transient internal pools, were not assayed.
RelativeAffinityfor CompetingSubstrates. The slope of the relationship between the ratio Vcm/VNH3 and the ratio [CH4]/[NH3] is the k c J K , , parameter used by Hyman and Wood [11] to compare the affinity ofN. europaea for ammonia and methane. For a single substrate/inhibitor ratio, this parameter was previously found to be 4.3 (in terms of NH3) [28]. Combining all the present data
Effects of Methane on Ammonium Oxidation Kinetics
217
for [CH4]/[NH3] less than 12, the slope is 0.004. This value is much lower than the previous result, which was derived from a single experiment at much lower methane concentration [28]. The present result, the average of several experiments, is the same value that was determined for the kcat/Km parameter for Nitrosomonas europaea [ 11 ]. Although the combined data yield a reasonable correlation coefficient (Fig. 5), significantly different slopes are obtained when each experiment is analyzed separately (not shown). Thus the ratio of substrate Concentrations is not necessarily an adequate empirical basis for prediction of relative reaction rates. The k c a t / K m parameter apparently is not constant and is of limited application in interpreting this interaction.
A Model for A m m o n i a Oxidation in the Presence o f Methane. The pattern of inhibition of ammonia oxidation in N. oceanus by methane may imply the presence of multiple active sites in the enzyme molecule. Very little is known of the geometry of the enzyme, ammonia monooxygenase, because it has proven extremely difficult to purify [18, 24-26]; little progress has been made since the earlier attempts of Watson et al. [30]. The behavior of whole cells may be more applicable than that of purified enzymes to predicting behavior in the environment, even if the underlying nature of the enzyme remains unknown. Adaptations of models that were originally developed to describe purified enZymes have been useful in other kinetic studies in marine systems, notably in nutrient uptake by phytoplankton [2]. The behavior of the whole cell system Under the conditions described here is consistent with the model of active site directed effectors of allosteric enzymes proposed by Smith et al. [19]. In this application, no distinction is made between activity observed in the whole cell and the activity that might be attributed solely to the enzyme (we cannot decipher the possible contribution of, for example, transport systems or feed back control mechanisms involving subsequent enzymes in the oxidative chain). In the model of Smith et al. [ 19], the inhibiting compound (ligand, L) is an analogue of the true substrate (S), and competition between L and S results in sigmoidal kinetics with respect to S. The model describes a cooperative enzyme that occurs in at least two active configurations (El and Ez), each with N active sites (N >- 2). El is the most abundant form of the enzyme and E2 is the least abundant. S binds preferentially to El (K~s -< Kzs) while L binds preferentially to E2 (K2L < K~L). Symbols and definitions are listed in Table 1. In order to compute the predicted VNH3 under conditions of substrate competition, several parameters must be measured or estimated. Some kinetic parameters for ammonia oxidation were measured directly, but others were estimated from the relationships implied by the model. The measured K,~ for ammonia (8 tzM) [28] was used as the Kts of the model, and Kzs (800 ~M) was chosen to reflect the higher affinity of the most abundant form of the enzyme for the primary substrate. Vmaxfor ammonia oxidation also had been measured directly (Table 1) [28]. The value used here is the one obtained from the experiments plotted in Fig. 8 (2.5 x 10 -14 M ) rather than the average value reported earlier (2.2 x 10 -14 M). IS] and [L] used in the model were the actual Substrate and ligand concentrations from the same experiments. In an earlier paper [28], a half saturation constant for inhibition (Ka.) was estimated from the change in Km for ammonia caused by inhibition by methane.
218
B. B. Ward
Table 1.
Terms and equations for substrate analogue model [19]
Symbol
Definition
Value calculated 2.5 x 10 -.4 variable variable 1 2 8
E, E~
initial velocity V .... m a x i m u m velocity (M cell -j h -t) substrate concentration ligand concentration monomeric (m = i) or polymeric (m -> 2) n u m b e r of enzyme configurations number of active sites more abundant form of enzyme less abundant form of enzyme
X
[E2]
0.01
go
Vm
Is] [L] m h n
[E,] Dissociation constants
Kts K2s KIL K2L
8 8 18 18
x x X X
10 -6 M 10 -4 M 10 -6 M 10 -7 M
Michaelis Menten equation for reaction velocity in the presence of a competitive inhibitor:
Vm[S]
Vo
K~s(1 +~[L] + [S]) Equation for reaction velocity in presence o f a ligand which is a substrate analog
117]:
vmFts](l
IS]
ELI)~
LK~,~\ +E-d+K-~,d
ts] (1
+xK-~
]Sl + tL] ~
+K~---~ K~,/ J
Vo = 1 +~--dls+K,e]
+X
1 +~s2s+KaL]
Relationships between variables for a ligand which is a substrate analog: K~s < 1; KIL > 1; X < 1
-
This was done to facilitate comparison with work by others in which the relationship between ammonia and methane oxidation had been interpreted as simple competitive inhibition. The K~ so estimated was 6.6 -+ 5.6 uM (N = 6). The uncertainty in this result arises from the necessity to subjectively select data from inhibition experiments which fall on the linear portion of the double reciprocal plots. As can be seen from Fig. 3, this is probably not appropriate, and the Ki so calculated is biased, if not meaningless. Because of conflicting results about the affinity of the enzyme for methane, and the inability to measure affinity constants for the competing substrate directly, it was necessary to estimate the value of K~L based on its relationship to Kls. K1L was estimated to be on the same order as, but slightly higher than K~s, indicating that the affinity of El for the competing substrate (L) is slightly less than its affinity for S. K2L was smaller than K~L, reflecting higher affinity of the least common form of the enzyme (E2) for the substrate analogue (L).
Effects of Methane on Ammonium Oxidation Kinetics
219 B
2,5
2.5 J=
a: 2.0 "6 1.5 E
-.
2.0
"o E
1.5
,e
~> 1.0
1.0
0.5
~
0.0 0.0
0.5 0.0
i
i
i
i
0,5
1.0
1.5
2.0
0.0
2.5
0.5
1.0
[NH3] 10 "s M
1.5
2.0
2,5
[NH3] IO'SM
D 2.5 2.0 ~.
1.2.
"6 E
0.8-
-6 1.5 E
~, 1.o 0.4.
~
~. n~
~
o.o" 0.0
I
i
=
i
i
1.0
2.0
3.0
4.0
5.0
[NH3] 10 .7 M
0.5
~!o
"
/
0.0 ---O-~o=,f ~ , 1.0 0.0 0.5
,
,
1.5
2.0
,
2.5
[NH3] 10"a M
Fig. 8, A. Comparison of experimental data (0 and O) and model results (A) for oxidation of NH3 in the presence of 3.5 #M CH4. Model i-esults were generated using the parameters given in Table 2 with [S] in the micromolar range and [L] = 3.5 #M. B. Model results generated using the Parameters given in Table 2, [S] in micromolar range, with varying [L]. O = 7 #M; [] = 4 #M; 9 3.5 uM; A = 2 #M; 9 = 0.1 #M. C. Model results generated using the parameters given in Table 2, except that [S] and ILl are at nanomolar levels, Symbols as in Fig. 8B. D. Model results generated Using parameters given in Table 2, except that affinity constants (Kls, K:s, KiL, K2L) have been reduced by 1,000-fold. [S] and [L] at nanomolar levels. Variable [L]: O = 100 nM; [] = 50 nM; 9 ~20nM;A= 10nM; 9
T h e k e y to p r o d u c i n g s i g m o i d a l k i n e t i c s is m u l t i p l e a c t i v e sites o n t h e e n z y m e m o l e c u l e . B e c a u s e r e l a t i v e l y l i t t l e is k n o w n a b o u t t h i s e n z y m e , t h e m o d e l w a s first r u n b y m a k i n g t h e s i m p l e s t a s s u m p t i o n s a b o u t t h e s y s t e m : a m o n o m e r i c e n z y m e ( m = 1), 2 e n z y m e c o n f i g u r a t i o n s (h = 2), e a c h w i t h 2 a c t i v e sites (n -~ 2). W h e n t h e m o d e l w a s r u n u s i n g IS] a n d [L] p r e v a i l i n g u n d e r e x p e r i m e n t a l COnditions, p r e d i c t e d V v s S p l o t s n e v e r e x h i b i t e d a s m u c h c u r v a t u r e i n t h e low [S] r e g i o n as d i d t h e a c t u a l e x p e r i m e n t a l d a t a . I n Fig. 8 A , it c a n b e s e e n t h a t r e a s o n a b l e fit o f t h e m o d e l t o t h e d a t a is o b t a i n e d w h e n n = 8. W i t h n = 8, t h e m o d e l r e a c t s to i n c r e a s i n g [L] b y i n c r e a s i n g s i g m o i d i c i t y (Fig. 8B), a n d at v e r y l o w [L], e s s e n t i a l l y n o r m a l h y p e r b o l i c k i n e t i c s r e s u l t . A p e r m u t a t i o n o f t h e m o d e l w i t h n = 4 f o r a p o l y m e r i c e n z y m e ( m >-- 2) d o e s n o t p r o d u c e sigmoidicity under the experimental conditions. Thus the configuration necessary to describe the experimental results requires that one monomeric enzyme POSsess s e v e r a l a c t i v e sites. Kinetic parameters which were derived from culture experiments at micro-
220
B.B. Ward
molar levels of [S] and [L], and which adequately explain the kinetic behavior of the system under these conditions, might not be appropriate to the behavior of the organisms under environmental conditions where IS] and [L] are likely to be in the nanomolar range. When the model is run using the kinetic parameters determined in culture but with [S] and [L] in the nanomolar range, no inhibitive effect of methane is seen and no curvature is evident in the V vs S plot of NH 3 oxidation (Fig. 8C). Linear kinetics result because the affinity constants derived from culture are about 1,000-fold higher than environmental IS] and [L]. Obviously, organisms with such low affinities would not be well adapted to exploit an environment with very low substrate concentrations. One possibility to reconcile field and laboratory observations is that organisms in culture are adapted to the high substrate concentrations routinely provided. The same organism might be able to produce enzymes with higher affinities when adapted to growing in a m o r e dilute environment. Environmental substrate concentrations in the range of
