Studies of the enzyme hexokinase. IV. The role of sulfhydryl groups

Studies of the enzyme hexokinase. IV. The role of sulfhydryl groups

ARCHIVES OF Studies BIOCHEMISTRY AND of the Enzyme PAOLO Prom BIOPHYSICS the Department 100, Hexokinase. FASELLA’ of Chemistry, IV. The Ro...

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ARCHIVES

OF

Studies

BIOCHEMISTRY

AND

of the Enzyme PAOLO

Prom

BIOPHYSICS

the Department

100,

Hexokinase. FASELLA’

of Chemistry,

IV. The Role of Sulfhydryl GORDON

AND

Massachusetts Received

(1963)

295-297

Institute

September

Groups

G. HAMMES

of Technology,

Cambridge,

Massachusetts

14, 1962

Titration of purified yeast hexokinase with p-mercuribenzoate showed the presence of six SH groups/lOO,OOO g. hexokinase. Simultaneous activity determinations indicated that in the presence of lo+ M glucose, all six SH groups could be titrated with essentially no loss of activity, although spontaneous denaturation quickly followed. In the absence of glucose and in the presence of 10-l M glucose 6-phosphate, enzymic activity decreased as the titration proceeded. This indicates that SH groups are not involved in the actual catalytic process, but probably help to stabilize the active enzyme conformation. INTRODUCTIOS

Previous papers in this series presented a general mechanism of action for yeast hexokinase derived through steady-state kinetic studies (1). The proposed mechanism involves formation of a quaternary intermediate composed of both substrates, a metal ion (usually Mg+2), and the enzyme. The data indicated a compulsory sequence of addition of substrates to the enzyme occurs, the established order being glucose, Mg ATP in the forward direction and with less certainty glucose C-phosphate, and Mg ADP in the reverse direction. The uncertainty for the reverse reaction derives from the inherent difficulty in uniquely fitting product inhibition data for such a complex reaction. In addition, the specific role of the metal ion in the catalytic process was investigated. Other workers have recently involving

proposed a a quaternary

similar mechanism intermediate, but

with random addition of substrates (2). In order to obtain information about the chemical nature of the catalytic process, we have determined the number of sulfhydryl groups present in hexokinase and the effect on the enzymic activity of block1 Present address: Tstituto di Chimica logica, University of Rome, Rome, Italy.

Bio295

ing them. Several attempts at such a study have been made previously, but the results obtained were somewhat ambiguous (3-5). The results which will be reported here indicate that sulfhydryl groups are not directly involved in the catalysis, but may be involved in maintaining the necessary protein conformation for enzymic activity. EXPERIMENTAL The yeast hexokinase used was a crystalline sample obtained from Dr. S. P. Colowick. The specific activity indicated t,he sample was 100% pure (1, 3). The procedure for the spectrophotometric titration of sulfhydryl groups with p-mercuribenzoate has been well established (6) and will not be discussed further here. The p-mercuribenzoate was obtained from Sigma Chemicals, the glucose B-phosphate from Calbiochem, and all other chemicals were reagent grade. The sulfhydryl groups were titrated at 25°C. in 0.2 fM phosphate buffer, pH 7. The following solutions were studied: 1. enzyme alone; 2. enzyme + lo+ M glucose; 3. enzyme + 10-l M glucose F-phosphate; and 4. enzyme + 8 ilf urea. The enzyme concentration was approximately 1 mg./ml. in all experiments. The concentrations of glucose and glucose 6-phosphate are such that over 90% of the enzyme should be bound (1). In all cases (except the urea solution), approximately one equivalent of the titrant was added, and as soon as equilibrium was reached the activity was

296

FASELLA

AND HAMMES ADP upon the titration-activity behavior were made; however, since the binding constants for the nucleotides with enzyme are less than lo3 M-l (l), nucleotide concentrations of low2 n/r or greater are required. The large absorption of the nucleotides at such high concentrations precluded obtaining significant results.

?O-

RESULTS

60:

50-

_ 5 t= 402 ae 30-

:jj

,

( I

TITRATED

,

,

2

,

(

,

3

SH GROUPS/IO’ HEXOKINASE

,

4

,

,

, !Flkli*

5 g

6 OF

FIG. 1. Enzymic activity versus titrated sulfhydryl groups: (a) 0 pH 7, 0.2 M phosphate buffer: (b) l same as (a) plus 0.1 M glucose g-phosphate; (c) n same as (a) plus 0.01 M glucose. The enzyme concentration was approximately 1 mg./ml. in all experiments. The time t = 0 is taken as the time at which the spectral changes were consistent with six SH groups having been titrated.

determined using the pH-stat assay previously described (1); after the assay was completed, the next equivalent of titrant was added. This procedure was repeated until all of the groups were titrated. The duration of the entire titration varied with the particular solution: for Soln. 1, the titration took approximately 1.5 hr.; for Soln. 2 about 3 hr.; and for Soln. 3 about 2 hr. The error in the activity determinations is about &57*. In order to obtain meaningful results, the activity measurements were carried out as quickly as possible to prevent errors arising from spontaneous enzyme denaturation. In addition, the activity of an untitrated enzyme solution was determined periodically and was used to correct the results for spontaneous denaturation. These corrections were usually less than a few per cent. For the titration in urea, a slight excess of titrant was added, and the changes in absorbancy with time were measured until equilibrium was reached. The titration in urea took about 30 min. for completion. Attempts to study the effect of ATP and

AND DISCUSSION

The titration in urea indicated the presence of six sulfhydryl groups/lOO,OOO g. protein. (Strictly speaking, this number of sulfhydryl groups must be regarded as a lower bound since the possibility exists that some groups may be masked to our titrating agent.) This is in accord with a preliminary amino acid analysis of hexokinase;2 however a performic acid oxidation was not performed prior to acid hydrolysis so that only a lower bound for the number of sulfhydryl groups was obtained. The rest of the data are summarized in Fig. 1. Note that titration of the enzyme alone and in the presence of glucose 6-phosphate causes a gradual loss in specific activity. On the other hand, all six groups can be titrated without loss of activity in the presence of glucose, although activity is lost after the sixth group has been titrated. It should be pointed out that addition of excess p-mercuribenzoate (more than six equivalents) to solutions not containing urea caused spectral changes; i.e., absorbancy increases, even after six groups had been titrated. However, the spectral changes occurred over the whole region from 220 to 350 rnM and undoubtedly represent protein denaturation. As a consequence of this, only the titration in urea, carried out in duplicate, establishes the absolute number of sulfhydryl groups present. The interpretation of these results seems quite clear. Since all of the sulfhydryl groups can be titrated in the presence of glucose with essentially no loss in activity, they are obviously not involved in the actual catalysis. However, the fact that inactivation occurs readily during the titrations in the absence of glucose and after the sulfhydryl groups are titrated in the presence of glucose indicates that these groups are probably involved in maintaining the active protein conformation. 2 H. Van Vunakis, private communication.

ENZYME

HEXOKINASE.

As pointed out by Boyer (6) and by Kaper and Houwing (7) among others, such a conclusion is not unequivocal since the presence of the p-mercuribenzoate radicals on the enzyme may be sufficient to disrupt the protein structure. Similar conclusions have been reached independently by S. P. Colowick and A. Kaji (private communication).

297

IV

Since we have successfully titrated these groups with very little loss in enzymic activity, this suggestion does not seem tenable. The two additional groups found by Barnard and Ramel (under experimental conditions different from ours) might conceivably be involved in the active site; however it would be unusual for the least reactive sulfhydryl groups to be locat,ed at the active site. REFERENCES G. G., AND KOCHAVI, D., J. dnz. Chew Sot. 84, 2069, 2073, 2076 (1962). FROMM, H. J., AND ZE~E, V., J. Biol. Chem. 237, 3027 (1962). BERGER, L., STEIN, M., COLOWICK, S. P., AND CORI,C. F., J. Gen. Physiol. 29, 379 (1946). DIXON, M., AND ~EEDIIAM, D. M., X&m 168, 432 (1946). BAILEY, K., AND WEBB, E. C., Biochem J. 42, 64 (1948). BOYER, P. D., “The Enzymes,” Vol. 1, p. 511. Academic Press, New York, 1959. KAPER, J. M., AND HOUWING, C., Arch. Bio-

1. HAMMES,

ACKNOWLEDGMENTS We are greatly indebted to Dr. S. P. Colowick for furnishing us with crystalline hexokinase and for allowing us access to his experimental results before publication. This work was supported by a grant from the National Institutes of Health (RG 7803). ADDENDUM After this work was submitted for publication, a brief note concerning the sulfhydryl groups in hexokinase appeared (8). Quite unreactive sulfhydryl groups were found, contrary to our observations, and it was suggested that sulfhydryl groups are necessary for the catalytic activity.

2. 3.

4. 5.

6. 7.

chew.. Biophys. 97, 449 (1962). E. A., AND &MEL, 84, 721 (1962).

8. BARNARD,

-4., Riochem. J.