J. ilfol.

Biol.

(1977)

114, 267-279

Further Investigations of the Transient Kinetics of Alcohol Oxidation Catalysed by Horse Liver Alcohol Dehydrogenase ANTONIO

BAICI AND PIER LCIGI LUISI

Eidgeniissische Technische Hochschule Ziirich Technisch-Chemisches Laboratorium, Universitdtstrasse CH-8092 Ziirich, Switxprland (Heceived

21 Decem,ber 1976, and in revi.&

j’orttl 10 April

6

1977)

Due to the controversy over the half-of-the-sit,es reactivit,y of llorsc liver alcohol u-c have re-investigated the dehydrogenase during benzyl alcohol oxidation. transient kinetics, stoichiometry and rate paramet-ers over a wide range of substrate concentrations (0.05 mM to 40 mM) at pH 7.0 and 8.5 and using newly determined extinction coefficient,s. Data were elaborated by computer analysis in order to separate the initial rapid step (burst) from t)he whole time-course of the reaction. It has been found that: (1) the dependence of the burst amplitude upon benzyl alcohol concentrat’ion is distinctly biphasic. In the range from is rather insensitive to 0.05 mM up to approximately 1 IIIM the burst amplitude changes in alcohol concentration and corresponds to 5091; of the active sites of the enzyme; for alcohol concentrations greater than 1 InM this amplitude increases and reaches a value of approximately 9070 when benzyl alcohol is 40 IIIM. (2) The steady-state initial rate is also biphasic with respect to alcohol concentration, indicative of substrate inhibit,ion, whicll begins in the concentration range at which deviation from the half-burst also appea,rs. In other words, burst amplitudes larger than 50qh are concomitant with inhibition of t,he rate of enzyme turnover. (3) In the presence of isobut-yramide the burst is larger than 50% for the whole range of concentration of the substrate and ext a closer c*orrespondcnce lctwten the, theoret’ical and the experimental st,arting points gencrally prrmit,s a higher degrcacx of reliability in the evaluation of the burst) amplibude.) Reactions have been investigat,ed OVPI a large range of concentrat,ions of subst,ratt, in order to cover the whole range of substrate concentrat,ions thus far reported in the literature. We will show in fact that, one major cause for discrepancy lies in the different concentrations used by the va,rious groups. This paper is restricted to the problem of a,lcohol oxida,tion, and constitutes also a results from this laboratory partial reassessment of some of the prcviou c:experimenta, (Luisi & Bignetti, 1974). Criticism of the half-of-the-sites reactivity on the side of benzaldehyde reduction has heen raised by Hadorn et ml. (1975). Tatcmoto (1975). and Kvassman & Pet,t,ersson (1976). This is considered by work from other laboratories (Morris & Dunn, 1976; Bernhard et al., unpublished data).

2. Materials and Methods All experiments were carried out wit.11 tlw pure EE isoenzyme prepared by the method of Lntstorf et al. (lOTO), starting from commercial preparations (Boehringer and Soehne. (I( fract,ions wwc pooled, concnntrxted Mannheim, Germany). The EE isor.nz~lnr~-corltaillill~ by ultrafiltrat,ion on Amicon Incmbranos (UM- 10) to a final concn of abont, 10 mg protein: csontaining Ifi?:, (I-/V) methanol/IO ml, and crystallized by dialysing agaitlst, a sohltioll

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260

miw-phosphate buffer at pH 7.0. Prior to use, the crystals were dissolved and dialysed 6 times against, a loo-fold vol. of the appropriate buffer. Enzyme homogeneity was confirmed by starch gel electrophoresis and the bands were detected by activity staining according to Moser et al. (1968). The enzymatic acti\-ity. expressed as NAD-binding-site concentration, was determined with the pyrazole titration technique of Theorell & Ponrt,ani (1963), and concentration of enzyme is expressed in this paper as normalit’) (N), i.c%. twice the rnola,r concrntration. enzymatic: acti\-it.ies ranged between 9.5 and I ooo,,. (i,) Coenzyme,

substrates

and

other chemicals

NAD + and NADH were obtained as commercial l)reparat,ions from Sigma and were of the highest purity available and were used nithout further purification. The percentage of crrzymatically active NAD+ was cllecked by assay with excess ethanol and in the presenre of horse li\-er alcohol dehydrogmase and was never less t,han 99?& of the total sample. NADH purity, also checked ellz>,matically wit11 excess acetaldellyde, was about 980/,,. Renzyl alcohol was a Fluka product and was distilled shortly hefore use lmder reduced pressure. Ethy l- 1,l -d, alcohol (987; isotopic purit)y) \vas obt’ained from Merck, Sharp & Dohme, Canada. Buffer salt,s Lvere Merck products of the llighest grade of purity. Water doubly distilled from a quartz apparatus was used for t,he preparation of reagent and buffer solutions. Sodium phospllate buffer (0.05 37) \vas nscd for the experiments at pH 7.0 and glycine/KOH (0.1 M) at pH 8.5. Isobut,yramide was obtained from Aldrich and pl~rifird by sublimation hefor IIS~. (~3) Btopped-jlow

measurements

In t,his work, the stopped-flow apparatus was connected to a PDP-8 computer which t:nabled us to st,ore the experimental results on a paper tape for further analysis and to record curves on a x-y recorder. Data on paper tape (400 points for each experiment) were analysed by means of a Fortran program as follows: (and Fig. 1 illustrates a typical experiment) (1) the apparent rate constant, ,Qtr, of the first phase (burst) was determined by tile Guggenheim method as described by Frost K: Pearson (1965) applied to the inibial part of the curve (typically 3 to 4 half-times) using a linear regression. (2) By back ext,rapnlation for the 3 ms of the dead-time of the instrlunent (which was determined on the hasis of t,he reaction between ascorbat)e and 2,6-dichlorophenol indophenol), 1, the expcriment~al start of the reaction (as log,mV) was determilled and compared with the theoretical start (corresponding to the sum of the optical densities of all reactant species). Experiments which gave a discrepanoy worse t,han i 0.003 O.D. unit’s between the 2 starting points were discarded. (3) The &, and I valurs wer(\ used to calculate the amplitude (A) of t,he burst according to the eqktation :

whew dt is the time interval uscAd in the Gllggenheim procedure. (4) An exponential curve was t,hen built with the rate const,ant k,,, the amplit,ude A, and the starting point 1. This ralcnlated curve was superimposed on the experiment,al curve and the degree of coincidence between experimental and calculated points was checked. In this procedure the At valur was varied so a,s to give at the same time the best linear Guggenheim plot and the best, fit, between experimental and calculated curves. In all the examples given in Tables 2 and 3 t’hr, deviation between experimental and calculated curves at each point was lower than t’hr experimental error (approx. 0,002 O.D. unit,, or 2 mV in the actual readings) in the range of 3 to 4 half-times in experiments without isobutyramide and 5 to 6 half-times in experiments with isobutyramide. The major source of error in this procedure is the precision by which k,, is evaluated, and in particular, cllrves with a small signal-to-noise ratio give larger standard errors for k,,. As a rule we discarded curves for which a standard error greater tllan 10% in the (Guggenheim plot was obtained. Tile reproducibility of the results was found to be a greater problem than the acxcumry of the best fit, in t,he sense that experiments carried out consecl&vely could give ris(, to curves, each of them fitting wrll \vit,ll the calculated exponentials, but wit,11 burst amplitudes differing by as much as 1I)(),~. In order to ox-ercome tlria source’ of (‘rror. s(‘\ cxrnl c>sporiments (typically 5 to 10)

.I.

2iO

BAIC’I

;\NI)

I’.

TABLE

Extinction

at 328 tm

pH

Enzyme NAD NADH Enzyme. NAD Enzyme. NADH Enzyme.NADHGobutyremide

LUISI

1

cotfficients

9, pccies

Data in parentheses

I,.

210 84 5550 300 5760 5640

are those of Hadorn

pH 8.6

7.0 405 84 5550 680 6080 5430

(400) (83) (5550) (688) (6080) (5400)

N-‘cm-l M-%I-’ %I ‘cm -- 1 N-lCIN1

~-‘cm~’ ~-km-~

et cd. (1975).

were carried out under exactly the same conditions (i.c. for oath set of cotlcentrations) and the mean value is reported in our results. All measurements were done under limiting conditions of the enzyme i.e. the enzyme concentration being smaller than that of coenzyme and substrate, and having enzyme in one syringe and the other reactants in the second one. All measurements were performed at 328 nm and 23 (f l)‘C. (d) Estinction

coe&ie&s

As pointed out by Hadorn et al. (1975), under saturating conditions of coenzyme and subst,rate, the difference in the extinction coefficients at 328 nm before and after reaction in the experiments without isobutyramide corresponds to the difference between enzyme. NADH and enzyme.NAD, and in experiments with isobutyramide it corresponds to the difference between enzyme*NADH*isobutyramide and enzyme.NAD. The extinction coefficient at 328 nm for the ternary enzyme*NADHGsobutyramide complex at pH 7.0 and 8.5 was measured by titration of the enzyme with NADH in the presence of saturating isobutyramide. The increase in absorbance was recorded with a Cary-14 spectrophotometer using the 0 to 0.1 absorbance slide-wire. The extinction coefficients of free enzyme, enzyme*NAD and enzyme.NADH were measured directly with the stopped-flow apparatus, by extrapolating the recorded trace to zero time, thus eliminating error possibly due to a blank reaction when coenzyme was present. Result*s are shown in Table 1, together with the extinction coefficients for the various species as reported in the literature. The values we found at pH 8.5 agree very well with those of Hadorn et al. (1975). We did not find any difference in the extinction coefficients of enzyme.NAD+isobutyramide and enzyme.NAD at either pH 7.0 or 8.5. In conclusion, the extinction coefficients used are 5460 and 5400 ti1-l cm-l for the transient reduction of NAD at pH 7.0 and 8.5, respectively, and 5340 and 4750 M-l cm-l at pH 7.0 and 8.5, respectively, for the same reaction carried out in the presence of isobutyramide. (e) Steady-state

measurements

The steady-state initial velocities for the benzyl alcohol oxidation reaction at pH 7.0 were determined on the stopped-flow apparatus with enzyme in the low7 N concentration range. The higher sensitivity gained with the computer attachment permitted us to was followed in the initial work at extremely small alcohol concentrations ; the reaction portion of its whole time-course (e.g. 0.5 to 5 s) and moreover the signal-to-noise ratio was improved by computer averaging of several successive experiments.

3. Results The

evaluation

of the burst

basis of the extrapolation correcting for the factor

to

amplitude

t = 0 from

was carried the zero-order

out in our previous steady-state

work

trace,

on the

and then

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(Gutfreund, 1975) ; k, and k, as a first approximation, having been set equal to the steady-state initial velocity and to the apparent rate of the burst, respectively. The ratio r/E,, where r is the burst amplitude corrected for the factor f, and E, is t’he total enzyme concentration, was taken as a measure of t,he fraction of enzyme sites which are involved in the rapid initial phase. One can argue that this method of correction is not very valid, because the enzyme does not always obey a Theorell-Chance mechanism, and because the values of k, and k, cannot be reliably evaluated under the actual experimental conditions. For this reason, we have now adopted an alternative method for calculation of the burst, amplitude. According to this procedure, the value of v is obtained by computer analysis without any other restriction than imposing a best fit with a single exponential to the first portion of the experimental kinetic curve. Figure 1 shows a typical example of this procedure. One may argue that our results are biased by the restriction that the exponential approach t,o the steady-state must follow first-order kinetics. Indeed, mechanisms can be proposed, which would demand tha’t the burst rate is composed of two or more chemical steps. On the other hand, we purposely did not want to impose, a priori, any kinetic model for processing the data. and indeed our procedure corresponds to t’he minimum possible set of external restrictions. The fact that in all cases a good fit. bet,ween experimental and calculated data is obtained over several half-times, suggests that our approximabion is basically correct, i.e. the burst process appears t,o be governed mostly by a single exponential decay.

FIG. 1. A typical stopped-flow trace for benzyl alcohol oxidation at pH 7.0. Experimental conditions are (concns after mixing) : enzyme (17.5 pi), benzyl alcohol (0.5 IIIM), NBD (1.1 mw). The recorded trace (-) is shown together with t,he calculated exponential ( -). The amplitude of the burst in this experiment corresponds to 8.9 ~LN, and the equilibrium of the reaction (upper trace parallel to the time axis) corresponds to 14.2 PN-NADH produced.

272

.I.

H.11(‘1

:1x1>

I’.

I,. l,L.ISI

Another objection to our proccdurc~ III:L~ lx that, t tub Guggenheim analysis is tlot the most accura.tt: WX~ to evaluatcx rilt(* ~oust.ant~. Rcwgnizing t,llis. 1st’ Ilad dwithcl to use the rate con&ant obtained /:ia that Guggenhcitn analysis onl~~ as ELI) initial vaIu(b for a successive b&-fit computat,ion. However, this second step appeared unnecessar? since the difference between t,he experimental resu1t.s and t.he theoretical (‘urvt’ obtained via the Guggenheim procedure IVBS smaller at8 each point) than the wpwimental uncertainty over several half-times. As already mentioned, most of the data obtained in this paper are based on cxtinction coefficients which have been revised or redetermined witch greater accuracy. They are reported in Table 1. Let us consider first the experiments at pH 7.0. Table 2 reports data pertaining to the oxidation of benzyl alcohol and deuteroethanol under various conditions, while Figure 2 shows a double reciprocal plot for the case of benzyl alcohol. Figure 2 (curve A) shows the influence of the substrate concentration upon the burst amplitude; curve B shows the influence of the substrate concentration upon the initial velocity of the process following the burst, a,s determined from the same experiments as in for curve A; and curve C reports the dependence of the steady-state initial velocity as determined by a parallel set of cxperimenbs with enzymr~ in the 1OV7 N concentration range. There are a few relevant observations to be made from Figure 2. First of all: the double reciprocal p1ot.s are markedly biphasic. In curve A the amplitude of the burst is rather insensitive to changes in substrate concentration in the range 0.05 mM to about 1 mM> and gives a n/E, value of 0.51 by extrapolation to infinite substrate

TABLET Benxyl alcohol and deuteroethanol

Benzylalcohol 0.05 0.10 0.30 0.50 1.00 2-01 5.02 20.1 40.2 0.50 1.00 5.00 40.0 -

-

Concentration (mix) Douteroethanol lsobutyramitle _-

_--0.52 1.07 2.15 21.5 1.07 21.5

100 100 100 100

100 100

oxidation

at pH 7-O

k,,(s - 1)

I’orcont~age burst (a/E,) x 100

13.7 12.3 16.6 16.7 15.2 15.5 9.5 13.0 I o-o 9.7 U.X 10.4 IO.4 5.3 5.5 7.5 23.7 0.7 3.8

454 48.8 50.6 50.8 52.4 61.0 ‘is.0 Xl.9 9o.L’ 62.3 75!) 87.3 00.9 54.2 67.7 73.0 X6.9 77.3 93.4

In all experiments NAD wras 1.1 nxw, and enzyme concentration ranged between 15 and 20 pN; t = 23 (i l)“C, Na phosphate buffer, 0.06 M. Each value is the average of 5 to 10 experiments.

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273

KINETICS

I.6

I.0

0.8

0.6 I 5

I 20

I 15

I IO l/[Benzyl

alcohol]

,I "

1 30

I 40

I -1 50

(m-’

Fra. 2. Double reciprocal plots for benzyl alcohol oxidation at pH 7.0. Influence of the substrak concentration upon: the r/E, values (curve A); the initial velocity of the process following the burst, as measured in the same experiments as (A) (curve B); true steady-state initial velocity as determined with catalytic amounts of enzyme (curve C), in these experiments enzyme was 0.17 /*N and NAD 0.9 max. The ordinate relative to curve a is on the left, that relative to curves B and C is on the right. Each point is the average of 5 to 10 experiments. For other details SW also Table 2. TJ is initial velocity ([substrate]/s).

concentration. At substrate concentrations larger than 1 II1M, tfhe T/~,, value begins to increase markedly. The initial velocity also has biphasic behaviour which is indicative of substrate inhibition. Inhibition by high alcohol concentration has been reported earlier, e.g. by Meier (1975) and Tatemoto (1975) for benzyl alcohol at pH 8.5, and by Shore & Theorell(1966) for ethanol at pH 7.0. In our case, inhibition is observed not only in the typical steady-state experiments. but also (Fig. 2(curve B)) under the conditions of the stopped-flow experiments (relat’ively high enzyme concentration). Note that the extrapolated value for the initial velocity is lower in the stopped-flow experiments (approx. Is- ‘) than under real steadystate conditions. Extrapolation to infinite substrate concentration gives a value of 1.8 s.- l for the turnover number obtained with dilute enzyme, which is close to the value of 2.0 s-l already published by Pietruszko et al. (1973), and somewhat greater t,han the value of 0.95 s-l that we found earlier with the stopped-flow method using larger enzyme concentrations (Luisi & Bignetti, 1974). In this particular reaction the difference in turnover numbers noted between the stopped-flow value and the classical steady-stabe value is certainly an interesting aspect of horse liver alcohol dehydrogenase kinetics. For a possible explanation, one may invoke the presence of a chemical

274

.4. B.AICI

.4iXD

I’. L.

LUIST

st,ep between the burst phase and bhcb zero-order steady-st,ate rate. In part,icular, if a protein conformational rhangc,. or ii substratcx dissociation stc,p (involving no optical den&y change) mixes with the zero order-rate after the burst, a lolver turnover number may be obta,ined with large (anzyme concentrations (in the extreme case this mechanism would result in a lag phase after the burst, and prior to the zero-order steady-state trace). Also explanations which are more in line wit’h the half-of-the-sites mechanism can be proposed. In particular, mechanisms can be devised, which would give, for the initial velocity following t’he stopped-flow burst, values one half as great as those obtained under classical steady-state conditions (or, equivalently, one must assume that only one half of the enzyme sites are a.ctive under the stopped-flow conditions in order to get numerically the same rate value). More simple interpretations can also be suggested ; for example, that classical kinetic steady-state approximations do not hold for our system under high enzyme concentrations. In principle, one might interpret the difference by invoking enzyme aggregation at the relatively high enzyme concentrations needed with the stopped-flow technique. However, to the best of our knowledge there is no indication of such a phenomenon for this enzyme. At this time we do not feel inclined to advance any particular one of these explanations for the difference in the turnover numbers. The most import,ant observation from Figure 2 is that the biphasicity obtained in the double reciproca,l plots of the burst amplitude parallels the biphasicity obtained by following the initial velocities. In other words, burst amplitudes larger than 50% begin to appear only at substrate concentrations corresponding to substrate inhibition. These results, while confirming the existence of the half-burst in the oxidation of benzyl alcohol which we have already published using a more approximate method (Luisi $ Bignetti, 1974), also require a, correction of some of the data presented in that earlier work. In fact in that paper, 500A, 1mrst, amplitudes were reported also for benzyl alcohol concentrat,ions greater t,han 1 to 2 mM, in contrast, with the present work. In the previous paper, we described stopped-flow experiments for the oxidation of This compound forms a benzyl alcohol (1 to 2 mM) in the presence of isobutyramide. tight ternary complex with NADH, and therefore it has been employed to stop reaction after the single transient production of NADH. Using the same extinction coefficient as for free NADH, we found that the reaction stops after production of NADH corresponding to half of the enzyme active-sites concentration (albeit noticing that under conditions in which the enzyme was not premixed with coenzyme the amplitude tended to increase). Experiments carried out in the present work over a wide range of substrate concentrations do not substantiate this earlier finding (see Table 2), because a burst amplitude greater than 5Oyh is obtained over the whole concentration range for substrate. The different results obtained in the earlier work are due with all likelihood to the interplay of two effects : the use of a larger extinction coefficient, and the different pre-mixing used. In fact, in the previous work the experiments with isobutyramide were performed with enzyme and coenzyme premixed, which makes the determination of the actual start more difficult, due to the blank reaction. Data pertaining to the oxidation of deuteroethanol at pH 7.0 are presented in Table 2. The steady-state initial velocity, as for benzyl alcohol, shows substrate inhibition, as reported by Shore & Theorell (1966), which is manifested at concentrations greater than about 10 mM. The burst amplitude, however, does not show any

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‘75

biphasicity in the concentration range investigated. and extrapolates to a value near to 9O”;b burst, when data are plotted in a double reciprocal plot as in Figure 2. The same value is obtained in the experiments with isobutyramide. Corresponding fullburst behaviour has already been shown on the side of acetaldehyde reduction (Luisi & Favilla, 1972). Let us now turn our attention to the data with benzyl alcohol at pH 8.5. As already mentioned, this pH has been used by Hadorn et al. (1975) and Tatemoto (1975), who could not detect any half-of-the-sites reactivity. it is important in this regard to bear in mind that Hadorn et al. only present data for experiments in the presence of isobutyramide (no multistep turnover under normal conditions) ; and Tatemoto’s data are derived by measuring the fluorescence of NADH product. Table 3 shows that the data for benzyl alcohol at pH 8.5 basically conform to the data at pH 7.0. In the substrate concentrationrange between 0.05 mM and 2 mM, the burst’amplitude is within the range 49% to 620/ (note that between 0.40 mM and 2.0 rnM> it does not exceed 5O’j,; of the enzyme sites) ; above 2 mM, there is a sharp increase of n/E, and this change in behaviour takes place in the concentration range which, as shown by Meier (1975), brings about substrate inhibition at the steady-st’attt initial velocity. In this case? as for pH 7.0, experiments with isobutyramide produce considerably larger burst amplitudes. Studies with ethanol emphasize again that the half-of-the-sites reactivity seems to be a peculiar property only of the aromatic substrate. In all cases, t’he presence of the inhibitor isobutyramide brings the burst1 amplitude to the same upper level of approximately 90%. At pH 7.0 with our enzyme concentration (approx. 20 pN), experiments at substrate concentrat,ions lower than 0.1 rnB[ do not produce any slow step after the burst phase. Such experiments might therefore be considered as single turnover experiments, although the initial substrate concent’ration is larger than the enzyme concentration. This feature is dependent upon t,he relative values of TABLE

3

Benzyl alcohol oxidat%ou at pH 84

Benzyl

Concentration (mM) alcohol Isobutyramide

0.06 0.07 0.10 0.20 0.29 0.40 0.50 1.01 2.03 10.0 40.0 0.10 0.20 1.01 40.4

-

100 100 100 100

In all experiments the NAD concentration 16 and 20 piv; t = 23 (&l)“C, glycine/KOH experiments.

k,,(s

‘)

9.0 15.1) 14.9 14.0 18.X 20.1 16.4 20.4 22.1 17.4 12.1 8.0 10.0 12..5 13.H

Percentage burst (n/E”) x 100 57.4 53.6 57.8 61.4 61.9 50.0 50.9 48.3 53.1 66.6 87.7 77.4 74.3 80.5 94.0

was 1.1 m&x, cxnzymc concentration ranged between buffer, 0.1 M. Each value is t,he average of 5 to 10

278

the equilibrium phase.

.I.

H.ilC’J

.\NI)

constant)s for alcohol oxidat’ion

I’.

I,.

I,UISJ

in the burst phase and in t,he t,urnovclr*

Finally let us consider the data rclativo to thch rate of the burst. As already mentioned, the value of k,, has been obtained by the Guggenheim procedure and must be considered as an apparelzt rate constant (since mechanistically it may result from more than one chemical process). The k,,. values for benzyl a,lcohol oxidation at pH 7.0 trend to a limiting value of approximately 16 s-l in the non-inhibitory concentration range of substrate. At pH 8.5 a more marked dependence upon substrate concentration seems present, although with some scattering, with a maximal extrapolated value of ktP, at infinite benzyl alcohol concent&ion, of about 20 s-l. In both cases (pH 7-O and 8.5) a slight inhibition effect of high concentrations of substrat’e seems to be present. In the presence of isobutyramide, at both pH values, k,, is slightly decreased with respect to its maximal value, i.e. 9.8 s-l at pH 7.0 (McFarland & Chu. 1975, report a value of 10.5 5 1.6 s-l), and 13.5 se1 at pH 8.5 (Hadorn ef al.. 1975. report for t,his constant values between 7.4 and 11.4 s-l in thn concentration range of benzyl of deuteroethanol alcohol from 0.1 mM to 3.8 II1M). Finally, the lc,, for the oxidation shows a marked dependence upon the alcohol concentration and extrapolates to a value of about 25 s-l, which corresponds to the value given in the literature (Short & Gutfreund, 1970). Here also the addition of isobutyramide significantly decreases the value of k,,. Concerning the evaluation of the amplitude of the reaction in the presence of isobutyramide, Hadorn et al. (1975) pointed out that the use of the Guggenheim plot is not correct because the amount’ of enzyme in an enzyme.NAD*isobutyramide complex is not known. However Hadorn et al. (1975) found very similar values for the extinction coefficients (683 and 630 N -’ cm-l for cnzyme.NAD and enzyme. NADisobutyramide, respectively). Thr error due to using 630 instead of 683 N-I in the cvaluat,ion of the burst is about l’)l, which is well below t,hc precision of cnlrl a given experiment’.

4. Discussion The data presented in this paper show that half-of-the-sites reactivity is a characteristic feature of liver alcohol dehydrogenase during benzyl alcohol oxidation in the non-inhibitory concentration range of substrat,e. The question raised in our previous work, of whether this feature could be extended to conditions other than pH 7: is answered positively. Furthermore, bhe fact that burst amplitudes larger than 50% are obtained only in the inhibitory concenbration range of substrate seems to suggest that a loss of the half-of-the-sites stoichiomet,ry is connected with a decreased enzyme turnover activity and with depressed rates of the burst. In the presence of isobutyramide, contrary to our previous observation, burst, a,mplitudes larger than 50% of the enzyme concentration can be obtained. In view of the data presented in this work, let us first examine the criticism of the half-of-the-sites reactivity during benzyl alcohol oxidation at pH 7.0. Shore et al. (1975) found a burst greater than 75% with 50 mm-benzyl alcohol. This is in agreement with our data: at a comparable substrate concent,rat’ion (see Fig. 2 (curve B) and Table 2) we obtain approximately SO); burst’. To the best of our knowledge, these authors report no stopped-flow experiment following the benzyl alcohol oxidation for the non-inhibitory concentration range of substrate. Concerning the oxidation of ethanol,

ALCOHOL

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27;

Shore 6 Gutfreund (1970) reported a full burst when operating with 100 miw-substrate concentration. Our data are in substantial agreement with this finding. Let us turn now to the criticism presented in the literature of the half-of-the-sites reactivity of horse liver alcohol dehydrogenase with benzyl alcohol as substrat*e. considering the experiments at pH 8.5 carried out by different authors. Tatemoto (1975) measured the burst amplitude using benzyl alcohol and ethanol over a wide concentration range by following the fluorescence signal of the NADH produced. He reported burst data with double reciprocal plots similar bo those of Figure 2. While for ethanol he obtained a practically full burst, a significantly lowet figure was obtained in the case of benzyl alcohol. This value appears, however, to be larger than our 500/, value. It is difficult to explain precisely the origin of this difference, because Tatemoto’s and our data are not directly comparable, since Tatemoto was able to work with enzyme and alcohol concentrations much IOWCI than ours due to the greater sensitivity of the fluorimetric technique. It is likely that a major source of discrepancy lies in the evaluation of the concentration of NADH produced. In this regard one should notice that the fluorimetric method, despite the higher sensitivity, does not permit a direct determination of concentration changes. *4 previous calibration is necessary, and the choice of the calibration standard ix not an easy matter (e.g., the binary enzyme.KADH complex would be inadequate in this case because the ternary complex between enzyme, NADH and alcohol shows less fluorescence than enzyme.NADH). Tatemoto argued, on the basis of fluorescence data only, that the incomplete burst amplitude does not reflect the lack of saturation of the enzyme sites by NSDH product, but only the lower fluorescence of the abortive t’ernary complex. However, our data show directly by monitoring absorption changes that. at least under our conditions. an incomplete burst is obtained. This seems to rule out Tatemoto’s interpretation as being the sole explanation for the incomplete burst observed during benzyl alcohol oxidation. Let us now consider the criticism raised by Hadorn et al. (1975). These authors also worked at pH 8.5 with benzyl alcohol. They only reported data in the presence of isobutyramide, so that a detailed comparison wit,h our multistep experiments is not possible. As far as the experiments with isobutyramide are concerned, our present results are in substantial agreement’ with those of Hadorn et al., both concerning the amplitude and the rate of the reaction. In conclusion, we believe that the data presented in this paper settle most of the controversy concerning the burst amplitude during alcohol oxidation by horse liver alcohol dehydrogenase. We believe that it is established that: (i) the burst amplitude for benzyl alcohol corresponds to one half of the concentra,tion of the sites in the non-inhibitory substrate concentration range at pH 7.0 and 8.5; (ii) in the presence of isobubyramide or with inhibitory concentrations of benzyl alcohol, the burst amplitude tends towards loo:/,; (iii) the oxidation of ethanol does not obey a half-burst mechanism. with or without inhibitors. If the controversy concerning the data can be considered settled, the controversy concerning the interpretation of data will probably continue. The main question iti to what extent does the 50%, burst amplitude reflect a functional half-of-the-sites reactivity. The argument against this co-operative mechanism is that the 500/ burst, amplitude may be due to an interplay of rate constants. We grant that in principle such a non-co-operative kinetic mechanism could be in operation. However, we would like to point out, that any kinetic scheme for benzyl alcohol should account for the 19

27x

4.

BAICI

ANI)

P.

1~. LUISI

following facts all at the same time: (1) the n/E0 value is weakly dependent on substrate concentration in the whole non-inhibitory concentmtion range: (2) the n-/E, value increases only in the substrate concentration region in which the normal activity of the enzyme is inhibited; (3) the half-burst is obtained at pH 85 as well as at pH 7.0; (4) a full burst is obtained when ethanol is substituted for benzyl alcohol. An attempt to interpret these data in a non-co-operative way is being carried out at present by Dr Dutler and his colleagues (H. Dutler, personal communication). The half-of-the-sites mechanism proposed earlier should now be generalized to include the new data, in particular the obtaining of larger burst amplitudes accompanied by inhibition during the steady-stat’e rate. In this regard. consider that this inhibition has thus far been ascribed to the formation of the species E,(NADH, Ale),. In keeping with our half-of-the-sites mechanism, it could instead be ascribed to t’he formation of species IV (scheme 1) : E(NADH,Ald)

E(MAD,Alc) I E(NAD,Alc) I

E(NADH, -+

+I E(NAD,Alc) IT

-)

E(NADH,Alc) -‘t

E(NAD,Alc) III

E(NAD,Alc) IV 1 E(NADH,Alc)

scheme 1

I E(NADH,Ald). v

The latter, in the presence of a large concentration of substrate could be formed very rapidly following aldehyde-product dissociation. This hypothesis appears to be in agreement with recent steady-state investigations by Dubied & von Wartburg (1976). These authors found evidence that certain patterns of inhibition of liver alcohol dehydrogenase suggest the existence of enzyme species in which the binding of inhibitor at one subunit of the dimeric enzyme changes the kinetic properties of the adjacent subunit. These studies, however, do not refer to substrate inhibition, but to inhibition by abortive complexes of the type enzyme.NSDtrifluoroethanol. In our case, we must further assume that the alcohol molecule which is rapidly bound at the NADH site, while inhibiting NADH dissociation and therefore enzyme turnover, would induce reactivity in the neighboring, not yet reacted, subunit. In order to rationalize this point, we only need to postulate that species III, or any other enzyme species which is not completely filled, is not reactive, and that t.he reactivity in the burst process is resumed when the enzyme dimer is fully bound with subst’rate or substrate-like molecules. In other words, in the normal reaction pathway, reactivity of species II would be impaired by the fast dissociation of the aldehyde product (this rate being larger than the rate of hydride transfer) to yield the unreactive species 111. Before the next reaction, there must be NADH dissociation and binding of NAD plus alcohol. However, in the presence of excess alcohol, species IV would be formed very rapidly. thus inducing reactivity (formation of V) and burst amplitudes larger than SOY,;. Following the same argument we can explain t.he large burst obtained in the presence of isobutyramide: the very rapid filling of the free site of species ITT by isobutyramidc would induce the hydride transfer reaction at the other unreacted site.

ALCOHOL

DEHPDROGEKASE

TRANSIEKT

KINETICS

2i9

WI, are indebted to a number of colleagues for helpful discussions and critical reading of the manuscript. In particular, we would like to thank S. A. Bernhard (Eugene, Oregon), E. Bignetti (University of Parma, Italy), W. Bloch (Reed College, Portland, Oregon), A. Dubied (University of Bern), M. Dunn (Riverside, California), H. Dutler (ETH. Ziirich), J. McFarland (University of Wisconsin) and B. Straub (ETH, Ziirich). This research was supported by the Swiss Nat,ional Scirncca Fo~~ndatioll (3.3810.74). / REFEREnJCES Bernllard, S. A., Dunn, M. F., Luisi, P. L. & &hack, P. (1970). Biochemistry, 9, 1% 192. Dubied, A. & van Wartburg, J.-P. (1976). Ezperientia, 32, 767. Frost. A. A. & Pearson. R. G. (1965). In Kinetics alrd ,Tlechanism, p. 49, Wiley & Sons Inc.. New York, London. Gntfrcund, H. (1965). An Introduction to the #t&y of Erczymes, Blackwell Scientific Puhlications, Oxford. Hadorn, M., Jolm, V. A., Meier, F. K. & Dutler, H. (1975). Eur. ,I. Biochem. 54, 65-73. Kvassman. .J. & Pettersson, G. (1976). Eur. J. Biochem. 69, 279-287. Lnisi, P. L. & Bignetti, E. (1974). J. 1MoZ. Biol. 88, 653-670. Luisi, P. L. & Favilla, R. (1972). Biochemistry, 11, 2303-2310. Lntst,orf, U. M., Schiirch, P. M. & van Wartburp.