Folia Mierobiol. 22, 92--97 (1977)

Streptococcal Extracellular NAD-Glycohydrolase. Optimal Temperature and Activation by Cysteine F. J. ZAHRADI~IK Institute of Hygiene and Epidemiology, 100 d2 Prague 10

t~eeeived July 15, 1976

ABSTRACT. Stroptpooocal extracellular NAD-glyeohydrolase (EC 3.2.2.5) exists in two distinct states with respect to the optimal reaction temperature. Bacteria produce the enzyme form with optimum activity at about 40 ~ Probably due to oxygen action, the enzyme is converted to a form with optimum activity at 30 ~ Compounds of the type of cysteine restore the initial state. The conversions are accompanied by enzyme activity fluctuations.

In studying metabolic inhibition in rabbit myocardium b y a streptococcus culture filtrate, Carlson et al. (1956) found an enzyme which catalyzes the hydrolytic cleavage of the N A D molecule at the site of the N-glycoside bond between nicotinamide and ribose (Carlson et al., 1957). The enzyme is released b y some streptococcus strains into the culture medium (Bernheimer et al., 1957; Lazarides and Bernheimer, 1957) and has been obtained in a highly purified (Pace etal., 1959) and a crystalline form (Fehrenbaeh, 1971). Its kinetic properties have been studied (Carlson et al., 1957; Petersen, 1961). The authors estimate its optimum temperature to be a b o u t 40 ~ Ayoub and Wannamaker (1963) described the effect of SH-group activators on the activity of this enzyme. The present paper shows the enzyme to exist in two forms, the properties of which are described. MATERIALS AND METHODS

.Microorganisms. Streptococcus pyogenes C 203 U from the collection of the Centre of Epidemiology and Microbiology was used. Preparation of enzyme. The supernatant fluid obtained after centrifuging (2200 g 2 • 30 rain) the strain culture grown for 18 h at 37 ~ in T o d d - H e w i t t broth was used as the enzyme sample; it was preserved b y adding 0.01~o merthiolate. Purification of the enzyme leads to considerable changes in the activity and in the kinetic properties as will be described elsewhere. Enzyme activity determination. The method was based on measuring the absorbance of the addition compound of I~AD with sodium cyanide at 340 nm (Colowick et al., 1951) according to Carlson etal. (1957). Addition of 1 M sodium cyanide solution to the reaction mixture stops the enzyme reaction practically instantaneously. The unit of enzyme activity is defined as 10 nmol N A D degraded at 37 ~ in 450 s in a reaction mixture composed of 0.1 ml of enzyme sample and 0.5 ml of I~AD

1977

STREPTOCOCCAL NAD-GLYCOHYDROLASE

93

solution in a concentration of 1 mg N A D / m l in 0.1 M phosphate buffer (pH 7.3) containing 0.1 ~ bovine albumin as protective colloid (Charlson et al., 1957). Compared with Carlson's work, the reaction mixture volume was reduced to onefifth. Since albumin was found to produce a noncontrollable effect on enzyme activity, it was replaced by polyvinylpyrrolidone (PVP, Nutritional Biochemicals, Cleveland) of equal concentration. All the solutions prepared except that of sodium cyanide were preserved b y adding 0.01% merthiolate. The individual constituents t e s t e d were added with glass micropipettes, which gave a reproducibility better

than :k 1%. Samples for testing NAD degradation were prepared b y adding 20 [~1 of variously diluted enzyme to 80 ~tl NAD solution. Enzyme dilutions were prepared b y using 0.1 M phosphate buffer (pH 7.3) containing 0.1% P V P and 0.01% merthiolate. The s a m e phosphate buffer was used for preparing a solution containing 1 mg NAD/ml. Immediately on adding the enzyme the test tube containing the reaction mixture w a s covered with a piece of polyethylene foil, sealed with a rubber stopper, shaken a n d placed into an incubating bath. After a predetermined incubation period, 0.6 ml 1 ~ NaON was added to the reaction mixture and the contents were stirred. B y measuring the absorbanee at 340 nm in a 1-cm cuvette a value designated E~ was obtained. Control samples in which all of the N A D was present in the form of the addition compound with cyanide were prepared b y adding into 80 ~tl of N A D solution first 0.6 ml of cyanide solution and after stirring, 20 ~tl of diluted enzyme sample. Practically no enzymic reaction takes place in this solution. The absorbanee value at 340 nm (the same cuvette) was designated E0. Distilled water served as the b l a n k in absorbance measurement. Activity a was calculated from the equation: a -~ 775.34(E0 -- E~) de -~ 775.34de. AE t h e relative enzyme dilution de being used. The numerical factor was calculated from calibration measurements and from the definition of enzyme activity units. :For each measurement of activity in each relative dilution of enzyme sample, Eo was determined at least twice; the standard deviation usually did not exceed ~= 1%. Samples for measuring Ei were prepared at least three times each. The stand a r d deviation for E~ was usually ~ 3 % . Since in measuring the activity in this TABLE I. Statistical e v a l u a t i o n of t h e effect of a l b u m i n a n d eysteine on e n z y m e b y S t u d e n t ' s t-test

Addition to dilution buffer mg/ml Bovine a l b u m i n

I

Probability Activity units

Cysteino

0

0

1557.5 ! 20.2

1

0

1735.8 4- 26.1

2

0

1911.7 ~ 13.1

4

0

1969.2 ::t: 28.6

0

2

2675.4 :t= 60.0

2

2

2693.6 :J: 61.8

of coincidence P

< 0.01 0.01 0.1--0.05 0.3

94

F. $. ZAHRADN~K

Vol. 22 2.5

1500, a

~,

4 (

~. aFt/as

1000

2.0

0

o

c

500

1.5

9, I

10

,

I

20

I

30

I

40

50 |.o

d

Fxa. 1. Time dependence of en. zyme activities. Cysteine-influenced activity (solid circles, aR) and activity measured in the absence of cysteine (large open circles, as} are plotted, along with the ratio of as/as (small open circles), d, Days.

w a y o~m computes the difference between two determined values, the magnitude of AE----E0 --E~, which in the optimal case lies between 0.2--0.45, must be taken into consideration. With AE values smaller than 0.2 the standard error rises steeply, while with AE values greater than 0.5 the dependence of the degraded quantity of N A D on the relative concentration of enzyme ce(ce : l/de) deviates from l i n e a r i t y which is due to the transltion of the reaction conditions from zero to first order. The standard error for the optimal measurement of activity is usually in the range of =[=1--3%. The standard errors calculated are shown in all the figures. At t h e points where the error is not indicated, the values obtained coincide exactly with the corresponding graphical representations. RESULTS

Effect of cysteine on enzyme activity Table I summarizes the calculated values of enzyme activity as measured under different conditions. The enzyme samples were diluted with 0.1 M phosphate buffer (pH 7.3) containing 1 mg PVP/ml and 0.01~o merthiolate, to which bovine albumin (Armour Pharmaceutical Co., London) and cysteine were added in different con. centrations. An enzyme sample stored for 8 months at + 4 ~ was used for the measurements. Reaction mixtures were incubated at 28 ~ for 450 s. Activities and standard errors were calculated from the slope of the linear part of the dependence of AE on the relative enzyme concentration. The results imply that enzyme activities were influenced b y albumin. The activation effect of albumin depends, at least in a certain range, on its concentration. In the presence of cysteine the partial activation effect of albumin disappears. Similarly to cysteine, thioglycollic acid, dithiothreltol (Calbiochem, Lucerne) and sodium dithionite activate the enzyme without any quantitative differences. E n z y m e activity was not affected b y hydrazine sulphate, ascorbic acid, ethylenediamine tetraaeetic acid, 8-hydroxyquinoline, cholesterol, merthiolate and P V P . Fe 2+, Ag +, H g 2+ and hydrogen peroxide were found to be effective inhibitors. These substances were tested in concentrations of up to 0.05 ~ (or 10 mg/ml in the case of PVP).

Long-term examination of enzyme activity The time dependence of a c t i v i t y was measured in the absence of cysteine (S, for stable activity) and in cysteine-activated samples (R, for reduced activity). For S a e -

1977

STREPTOCOCCAL NAD-GLYCOHYDROLASE qC SO i

LO :

0

-

m

~

--

30

20

10

i

o

J

I~

~e 3.S

3.0

/ 2.5

31,

3:3

31,

31S

FIG. 2. Temperature dependeneo of R a n d S activities (a) directly after cultivation. I n t h e ease o f R activity (solid circles), the en. zyme was activated b y 2 mg cysteino per ml buffer. I n the c a s e of" S activity (open circles), eysteino w a s n o t added to t h e buffer.

t i v i t y the sample was diluted with 0.1 • phosphate buffer (pH 7.3) containing 0.01% merthiolate and 0.1% PVP. For R activity 2 mg cysteine/ml was added t o the above buffer. Samples were incubated at 28 ~ for 450 s. The activity of enzyme stored at ~-4 ~ was followed for about 50 days. The result is demonstrated in Fig. 1. The measurements showed that the value of cysteine-enhanced activity is stable, Enzyme activity in the absence of cysteine, on the other hand, decreases and stabilizes at a limit value in a b o u t 1 month.

Temperature dependence of enzyme activity in sample measured directly after cultivation Samples were tested for both S and R activity at different temperatures. The enzyme was diluted so as to give an approximately equal decrease of substrate concentration at each temperature. The results of the measurement are in Fig. 2. The results of the measurements demonstrate that cysteine has an effect on t h e enzyme even directly after cultivation. However, it m a y be inferred from the temperature dependence curve of S activity t h a t the optimal temperature for the enzyme directly after it is produced b y bacteria is above 30 ~ perhaps up to 40 ~ The optimal temperature for the cysteine-actlvated enzyme was found to be above 40 ~ T ~ L E II. Calculated values of activation energy (Ea for hydrolysis, E ~ a s t f o r t h e d e n a t u r a t i o n l ~ i o n ~ E n z y m e sample Directly after oultivation, S Directly after cultivation, R 10 m o n t h s later, S 10 m o n t h s later, R

Ea 38.35 44.63 55.77 49.32

:J:: 0.96 4- 0.38 4- 1.34 4- 0.84

Edenat --187.7 --303.0 --93.3 --155.8

g- 17.0 4- 9.8 -4- 6.5 -}- 3.9

96

F. ft. ZAHRAD~:~K ~

Vol. 22

50

~0

30

20

=

l

j

I

10

35

/log a

3.0

2.5

3.1

3

3.3

34

3.5 103/T

FzG. 3. Temperature dependence of R and S activities in a sample s t o r e d for 10 months. Symbols as in Fig. 2.

Temperature dependence of enzyme activity in a lO-month sample An enzyme sample prepared directly from a bacterial culture was stored for 10 months at 4 ~ Traces of sediment were spun off at 2000 g for 10 min. The temperature dependence of enzyme activity was measured similarly as in the preceding experiment. The result of the measurements is demonstrated in Fig. 3. A clear difference from the measurements performed directly after cultivation is the finding of a temperature o p t i m u m at approximately 30 ~ for the non-cysteine activated sample. The activated sample, on the contrary, does not display any substantial difference in the optimal temperature. The calculated values for the activation energies (Ea) and activation energies of thermal disintegration of the enzyme molecule (Edenat) are summarized in Table II. The values given were calculated from the measurements shown in Figs. 2 and 3. Points in the close vicinity of the optimal temperatures were not used in the calculations. DISCUSSION

Streptococci produce the enzyme in the R form with a higher optimum temperature, as shown in Fig. 2. A change in the optimum temperature during storage of the enzyme probably indicates that the spatial structure of the enzyme undergoes a change, as shown in Fig. 3 Substances of the type of cysteine abolish this change and revert the enzyme to the state in which it is produced b y bacteria. This action is most probably associated with the formation of disulphide bridges via oxidation b y atmospheric oxygen. The character of the course of the an~as ratio in time (Fig. 1) suggests that a stepwise process m a y be involved. The unusual flatness of the peak of the curve for the temperature dependence of enzyme activity in the eysteine-nonactivated sample directly after cultivation (Fig. 2, circles) could be due to atmospheric oxidation under aerobic cultivation. The flatness of the peak could be accounted for b y a portion of molecules of the S form of the enzyme, whose development cannot be precluded under the present mode of cultivation. The oxidation alteration in enzyme structure m a y be considered to be complete in approximately one month. In this form the enzyme remains stable for a period

1977

STREPTOCOCCAL N A I ) - G L Y C O I ~ s

97

of several months without changing its properties. The high stability of the enzyme indicates that the role of proteases is limited. The relation between the two enzyme forms may be summed up under a working hypothesis as expressed b y the following model: [ R_enzyme I optimum temperature c. 40 ~ - - E d e n a t ~ 145 k J tool -1

~ oxidation reduction

S-enzyme optimum temperature c. 30 ~ --Edenat - - 93 k J mo1-1

Besides the low o p t i m u m temperature, an unusually low slope of the line for thermal disintegration, calculated at --93 k J reel -1, is characteristic for the S enzyme. Values reported for protein denaturation are usually in the range from --145 to --420 k J mol-L In the overall evaluation of the results, it is necessary to bear in mind the limited reproducibility of the measurements of optimum temperature and the individual activation energies. Nevertheless, the alterations observed are so pronounced t h a t oxidative changes in structure are highly probable. REFERENCES

AYOUB E. M., WA~r~AMAKER L. W. : A factor other t h a n streptococcal nicotinamide adenine dinucleotida~ which combines with a n t i b o d y to this enzyme. I t s production a n d effect on a n t i b o d y determinations. J. ImmunoL 90, 793, (1963). BERNHEIMER A. W., LAZARIDES P. D., WIL~O~ A. T. : Diphosphopyridine nucleotidaso as a n extracellular product of streptococcal growth a n d its possible relationship to loukotoxicity. J . J~xpa. •ed, 106, 27 (1957). C~LBO~ A. S., KELL~'BR A., BV.R~THEXMERA. W.: Selective inhibition b y preparations of streptococcal filtrates of the oxidative metabolism of mitochondria procured from r a b b i t myocardium. J . ExptJ. Med. 104, 577 (1950). CARLSON A. S., KELLNER A., BERNHEIMER A. W., ~REEMAN E. B.: A streptococcal enzyme t h a t acts specifically upon diphosphopyridine nucleotide: characterization of the enzyme a n d its separation from streptolysin 0. J. Exptl. Med. 106, 15 (1957). COLOWICI{ S. P., KApI~N N. O., CIOTTI M. M.: The raction of pyridino nucloctido with cyanide a n d its analytical use. J. Biol. Chem. 191,447 (1951). FEHRENBACH F. J.-" Reinigung u n d Kristallisation der NAD-Glykohydrolase aus C-Streptokokkon. Eur. J. Biochem. 18, 94 (1971). LAZ~aIDES P. D., BERNHEI~IER A. W.: Association of production of diphosphopyridino n u c l e o t i d a ~ with serological type of group A streptococcus. J . Bayer/el. 74, 412 (1957). PACE M. G., P~PP~.N~X~rER A. M.: A n immunochemical s t u d y of streptococcal diphosphopyridine nucle~tidase. J. ImmunoL 88, 83 (1959). PS~RS~.~T K. F.: U n t e r s u c h u n g e n fiber die Streptokokken Diphosphopyridinnueleotidasr II. Reaktious. kinetische Studion, Entwicldung yon Methoden zum E n z y m - u n d Antik6rper-Nachweis. Z. f . ttyg. 147, 357 (1961).