The active site of 6-phosphogluconate dehydrogenase

The active site of 6-phosphogluconate dehydrogenase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 189, No. 2, August, pp. 516-523, 1978 The Active Site of 6-Phosphogluconate A Phosphate MARIO RI...

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ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 189, No. 2, August, pp. 516-523, 1978

The Active

Site of 6-Phosphogluconate

A Phosphate

MARIO

RIPPA,

Binding

Dehydrogenase

Site and Its Surroundings’

MARCO SIGNORINI, TIZIANA FRANC0 DALLOCCHIO

BELLINI,

AND

Istituto

di Chimlca Biologica.

liniuersith.

Ferrara,

1tal.v

Received January 26. 1978; revised April 3, 1978 Tetrahedral anions hind to a phosphate binding site of 6phosphogluconate dehydrogenase from Can.dida utilis, inhibit the enzyme competitively with the 6-phosphogluconate, decrease the reactivity of the SH groups, and mimic the protective effect of 6-phosphoglu conat,e against some inactivating agents. The reaction of the enzyme with butanedione results in the inactivation of the enzyme associated with the modification of a single arginine residue per subunit. This arginine residue may be involved in the binding of the phosphate to the enzyme. Inactivation of the enzyme, upon reaction with permanganate, appears to be due to the oxidation to cysteic acid of a single cysleine residue per enzyme subunit. The reaction of the enz.yme with either periodate or hexachloroplatinate causes the loss of the catalytic activity. This inactivation, due to an affinity labeling, is correlated with the oxidation of two SH groups per subunit to an S-S bridge. Photoinactivation of the enzyme by pyridoxal5’-phosphate is also restricted to the active site of the enzyme. The lysine and the histidine residues involved in this photoinactivation should thus be in the vicinity of the phosphate binding site.

Many known enzymes have phosphate derivatives as a substrate, coenzyme, or effector. In most cases the phosphate group plays a critical role either in the binding to the enzyme, or in catalysis, or in both processes. In the last few years there have been many reports of the participation of arginine residues in the binding of phosphate to enzymes. This information, though interesting, is not sufficient to establish the mechanism of binding, since the chemical environment of the arginine residue must contribute to the binding process. Thus a study of other residues at the phosphate binding sites should provide interesting information on the structure of these domains. In this paper we report evidence for the involvement of an arginine residue in the binding of phosphate ions at the active site of 6-phosphogluconate dehydrogenase. Us’ This work was supported by Grant CT 7601313.04 from the Italian C.N.R. We thank Dr. C. A. Bartocci and F. Scandola of the Istituto Chimico of the University of Ferrara for many fruitful discussions.

MATERIALS

AND

METHODS

Crystalline 6.phosphogluconate dehydrogenase (6 phospho-n-gluconate:NADP+ oxidoreductase, decarboxylating, EC 1.1.1.44) from Candida utilis was prepared and assayed as previously reported 11). The enzyme has a molecular weight of 100,000 (1). The crystalline enzyme was freed from ammonium sulfate by gel filtration on Sephadex G-25 equilibrated with the buffer required in the subsequent experiment. The buffers used were: acetic acid-acetate, pH 6.0, Tri-HCl, pH 7.5, or boric acid-NaOH. pH 7.5; all buffers were 50 mM and contained 0.1 mu EDTA. 6Phosphogluconate (6PG),* NADP’, and DTNB were purchased from Sigma Chemical Co., St. Louis, MO. All other reagents were purchased from Carlo Erba, Milan, and were of analytical grade. All operations were carried out at room temperature, unless * Abbreviations used: 6PG, 6-phosphogluconate; DTNB, 5,5’-dithiobis(2-nitrobenzoate); PLP, pyridoxal 5’-phosphate, TEA, triethanolamine.

516 0003-9861/78/1892-0516$02.00/O Copyright 0 1978 hy Academic Press, Inc. All rights of reproduction in any form reserved

ing the technique of affinity labeling and two new enzyme inactivators, we detected the presence of several amino acid residues in close proximity of the phosphate binding site.

ACTIVE

SITE

OF 6-PHOSPHOGLUCONATE

DEHYDROGENASE

517

otherwise stated. The kinetics of the reaction between the enzyme and an inactivating agent was followed by diluting aliquots of the reaction mixture in acetate buffer at the desired time intervals and assaying them immediately for residual enzymatic activity. Titration of the sulfhydryl groups was carried out as described by Ellman (2). Amino acid analyses were carried out on the protein hydrolyzed at 110°C for 24 h in 5.7 N HCl, using the 4101 LKB amino acid analyzer. RESULTS

AND

DISCUSSION

Inhibition by Phosphate Ions and Their Analogs Both substrates, 6-phosphogluconate and NADP’, of 6-phosphogluconate dehydrogenase contain phosphate groups; the active site of the enzyme must therefore contain at least two separate binding sites for phosphate ions and inorganic phosphate should be an inhibitor of the enzyme. Indeed, inhibition by inorganic phosphate has been reported for the enzyme from Candida utilis (3) and from other sources (4-6). In order to obtain additional information on the phosphate binding site(s) of the enzyme, we have tested as enzyme inhibitors other anions which in their steric structure and electric charge can be considered as analogs of phosphate. Arsenate, sulfate, molybdate and tungstate, which have the same tetrahedral structure as phosphate, all inhibit the enzyme competitively with 6PG (Fig. 1) and noncompetitively with NADP’ (data not reported). The values of the inhibition constants, calculated from the experiments carried out at a fixed concentration of NADP’ are 26 mM for phosphate and arsenate and 16 mM, 14 mM, and 0.8 mM for sulfate, molybdate, and tungstate, respectively. These results suggest that these anions bind specifically at the site involved in the binding of the phosphate group of 6PG and not at the site for the binding of the phosphate group of NADP’. The action of phosphate analogs (see below) may thus be localized at the binding site of phosphate of 6PG. Other tetrahedral anions, such as permanganate, periodate, perchlorate, and chromate, inactivate the enzyme (see below) and there is evidence that they also bind to the binding site of phosphate. Anions with a planar structure, like borate,

FIG. 1. Inhibition of the enzyme by some tetrahedral anions. The reaction mixture contained 50 mM Tris buffer, pH 7.5, 0.1 no EDTA, 0.25 mM NADP’, 6.6 nM enzyme and 6PG at the concentrations indicated in the abscissa. Symbols: 0, no other addition; 0, in the presence of 20 mu phosphate or 20 mM arsenate; A, in the presence of 20 mM sulfate or 1 mM tungstate; 0, in the presence of 20 mu molybdate.

bicarbonate, and nitrate, do not inhibit the enzyme appreciably. The inhibition by tetrahedral anions is not due to ionic strength since sodium chloride, even at 0.1 M does not inhibit the enzyme. These experiments suggest that negative charge and tetrahedral structure are both. required for inhibition. Inactivation

by Butanedione

The reaction at pH 7.5 of 6-phosphogluconate dehydrogenase with butanedione results in the inactivation of the enzyme. The reaction follows pseudo first order kinetics until 90% inactivation is reached (Fig. 2). The presence of NADP’, 6PG, phosphate, or tetrahedral anions in the inactivation mixture causes a decrease in the inactivation rate. These experiments were carried out in borate buffer; when the borate buffer was replaced with TEA or Tris buffer, no inactivation was observed, providing further evidence for the formation of a product-stabilizing borate complex. The rate of inactivation is a function of the concentration of butanedione: plotting the logarithm of the reciprocal of the inactivation half-time against the logarithm of the concentration of butanedione (Fig. 3) a straight line is obtained, with a slope of

518

RIPPA

‘0 \

i ‘0 i

I

I 5

1 10

I

1

>

15 20 time (mlnl

lncubatlon

FIG. 2. Kinetics of inactivation of the enzyme by butanedione. A solution of enzyme (2 nmol of subunit/ml) in 50 mM borate buffer, pH 7.5, was treated with butanedione (final concentration 20 mM). Symbols: 0. no other addition; 0, in the presence of 0.7 mM 61’(;, 20 mM sulfate, or 20 mM molybdate; a, in the presence of 20 mM phosphate; A, in the presence of 0.2 nlM NADP’; q , in the presence of Tris buffer instead of borate buffer.

-IX.-

-1.0 ? /

z-1.2-

-

.p -u

,p'

1

0.2

The Reactivity Enzyme

-

I 0.L

(13-16) the inactivation could not be reversed. No reactivation was observed when the Sephadex column was equilibrated in borate buffer. Titration of the SH groups showed that the inactive enzyme has the same SH content of the untreated enzyme. Amino acid analyses showed that the inactivation can be correlated with the modification of only 2 out the 15 arginine residues present in each enzyme subunit. The kinetic analysis, however, indicates that the modification of only one of these two residues is correlated with the loss of enzyme activity. The specificity of butanedione for arginine residues (17-20), the effect of borate on both the inactivation and the reactivation processes and the protective effects of 6PG, NADP+ and tetrahedral anions, all point to the presence of an arginine residue, essential to the catalytic activity, at the active site of the enzyme. As has been proposed for other proteins (14, 20-26) the arginine residue involved in the inactivation by butanedione may be the binding site for the phosphate group of 6PG and also for the tetrahedral anions. The involvement of an arginine residue in the binding of the phosphate group of glucose 6-phosphate to a glucose 6-phosphate dehydrogenase has been recently reprted (27).

-

-

I

ET AL.

I

1

0.6

0.8

log[butanedlond

I

lmMi

FIG. 3. Determination of the order of the reaction between the enzyme and butanedione. A solution of enzyme (2 nmol of subunit/ml) in 50 mM borate buffer, pH 7.5, was treated with butanedione at the final concentrations indicated in the abscissa. The inactivation half-time 7 was calculated from semilogarithmic plots of activity against incubation time.

0.90. This means (7-12) that the inactivation is the result of the reaction with butanedione of a single amino acid residue per active site. Enzyme 90% inactivated by treatment with butanedione can recover up to 30% of its original activity after gel filtration through a column of Sephadex G-50 equilibrated with Tris buffer, pH 7.5. The extent of reactivation is small, in other cases

of

the SH Groups

of

the

The enzyme contains 4 cysteine residues per subunit (28) all titrable with DTNB even in the absence of denaturing agents. We have reported that the active site of the enzyme contains a cysteine residue close to a tyrosine residue (29) and that two SH groups are sufficiently close, in the threedimensional structure at the active site, to form a disulfide bridge (30). Titration of the SH groups with DTNB (Fig. 4) shows that the four SH groups have different rates of reaction with DTNB, indeed plotting the percentage of unreacted SH groups against the time of reaction four lines can be drawn (Fig. 5). If the titration of the SH groups is carried out in the presence of either 6PG or NADP+ the rate of reaction is drastically reduced (Fig, 4); the tetrahedral anions tested were found to mimic the substrate and the coenzyme in

ACTIVE

SITE

OF 6-PHOSPHOGLUCONATE

20 LO 60 a0 lncubatton

tlmelmln

4. Kinetics of the reaction between the enzyme and DTNB. A solution of enzyme (8.4 nmol of subunit/ml) in acetate buffer (50 mM, pH 6.0) was treated with DTNB (final concentration 0.02 mg/ml). Readings were made at 412 nm and the data obtained were converted to moles of SH groups titrated per subunit mole. Symbols: 0, no other addition; 0, in the presence of 6PG or molybdate; A, in the presence of NADP’; A, in the presence of phosphate or arseniate; q , in the presence of sulfate. The concentrations of substrate, coenzyme, and anions were the same of that reported in the legend of Fig. 2. FIG.

decreasing the rate of reaction. Chloride ions or planar ions, like borate or nitrate, have no effects on the reaction rate (data not shown). These results indicate that the binding of tetrahedral anions to the enzyme cause a change in the reactivity of the SH groups and suggest that at least some of these groups are near the phosphate binding site. If the same titration is carried out on the with butanedione, enzyme inactivated phosphate or 6PG are found to have no effect on the rate of titration. This suggests that the phosphate no longer binds to the same site in the modified enzyme and provides further support for the hypothesis that the phosphate binds to an arginine residue. The Reactivity of Histidine Residues Of the 6 histidine residues in each subunit of the enzyme, two are more reactive than the others toward diethylpyrocarbonate at pH 6.0 and at least one of these is located at the active site of the enzyme (31). The presence of 6PG in the reaction mixture decreases the reactivity of these residues and protects against the inactivation (31).

519

DEHYDROGENASE

We have now reinvestigated the inactivation of enzyme by diethylpyrocarbonate and seen that phosphate and other tetrahedral anions do not mimic the effect of 6PG on the reactivity of the histidine residues. Thus while tetrahedral anions are able to change the reactivity of SH groups they do not have the same effect on the reactivity of histidine residues. It may be postulated that these residues are out of the field of action of the bound tetrahedral anions and thus not very close to the phosphate binding site. The differences in effects of tetrahedral anions on the reactivity of two different types of residues decrease the possibility that gross conformational changes result from the binding of phosphate to the enzyme. Inactivation

by Phosphate Analogs

Tetrahedral anions bind to the phosphate binding site of the enzyme. In order to obtain additional information regarding that site we sought a tetrahedral anion that could bind to the phosphate binding site and then react with a nearby amino acid residue. Phosphate analogs like permanganate (32, 33) and ferrate (34) have been used as oxidizing agents specific for the phosphate binding site of other enzymes. The reaction at pH 6.0 between the enzyme and permanganate (0.01 mM) results in an almost instantaneous inactivation of the enzyme, but at 0°C and with lower concentrations of enzyme and permanganate the kinetics of the enzyme inactivation could

10 Incubatjon

20 tfme

io /mcnl

FIG. 5. Division of the SH groups in classes, according to their reactivity toward DTNB. The conditions are the same as in the legend of Fig. 4.

520

HIPPA

be followed (Fig. 6). The kinetics of inactivation by periodate and hexachloroplatinate at 20°C is reported in the same Figure. All inactivations follow a pseudo first order kinetics; the presence of 6PG, NADP’ and tetrahedral anions decrease the rate of inactivation. Hexachloroplatinate was tested on the assumption that the enzyme would also recognize the triangular face of an octahedral anion.

ET AL I

I

1

Kinetics and Stoichiometry of the Inactivation by Phosphate Analogs A more detailed kinetic analysis was carried out with periodate and hexachloroplaFIG. 7. Determination of the order of the reaction tinate. The rate of inactivation was found to be a function of the concentration of between the enzyme and periodate or hexachloroplainhibitor. A plot of the logarithm of the tinate. Solutions of enzyme (2 nmol of subunit/ml) in acetate buffer pH 6.0 were treated with the inactivatreciprocal of the inactivation half-time (T) ing agents at the final concentrations indicated in the against the logarithm of the concentration graph and the inactivation half-time r at each concenof the inhibitor yielded (Fig. 7) straight tration of the inactivation agent was calculated. Inaclines with slopes of 1. This means (7-12) tivating agent: periodate, upper line; hexachloroplatinthat inactivation is the result of the reaction ate, lower line of the inhibitor with a single amino acid residue per enzyme active site. A plot of r inactivation obeys saturation kinetics against the reciprocal of the concentration (35-37) providing evidence that the enzyme of the inhibitor (Fig. 8) shows that the and the inactivating agent (periodate or hexachloroplatinate) form a dissociable complex prior to the inactivation. Thus the inactivation must be due to an affinity labeling (37). From Fig. 8 it may be calculated that the Kiinact is 0.1 mu for periodate and 0.25 mM for hexachloroplatinate. Titration of the enzyme activity with less than stoichiometric amounts of inhibitors indicates (Fig. 9) that complete inactivation is correlated with the addition of only 3 mol of permanganate, 1.5 mol of periodate or 1 mol of hexachloroplatinate for each mole of enzyme subunit. The enzyme inactivated FIG. 6. Kinetics of inactivation of enzyme by oxiwith dizing phosphate analogs. A solution of enzyme (0.12, for the extent of 90% by treatment or hexachloroplatinate can renmol of subunit/ml) in acetate buffer (50 mM, pH 6.0) periodate cover in less than 20 mins up to the 70% of was treated with permanganate (final concentration 1.6 ,uM) at 0’ (left). A solution of enzyme (2 nmol of the original activity upon treatment with subunit/ml) in acetate buffer pH 6 was treated with 10 m&r mercaptoethanol in Tris buffer, pH periodate (final concentration 75 KM, center). A solu7.5. The hexachloroplatinate is an oxidizing tion of enzyme (2 mnol of subunit/ml) in Tris buffer, agent able to oxidize SH groups to disulfide pH 7.5, was treated with hexachloroplatinate (final bridge; indeed it is able to transform gluconcentration 5 pM, right). Symbols: 0, no other adtathione in oxidized glutathione, as dedition; 0, in the presence of 6PG or molybdate; A, in tected by glutathione reductase and the presence of phosphate or arsenate; 0, in the presNADPH. No recovery of activity was obence of NADP’. The concentrations of substrate, served on treatment with mercaptoethanol coenzyme and anions are the same as in the legend of Fig. 2. or borohydride after inactivation with per-

ACTIVE

SITE

OF 6PHOSPHOGLUCONATE

FIG. 8. Determination of the K,,.,, of periodate and hexachloroplatinate. Inactivating agent periodate (left), hexachloroplatinate (right). I

I

[Enzyme

I

I

subunlt]

FIG. 9. Titration of the catalytic activity of the enzyme with substoichiometric amounts of inactivating phosphate analogs. Solutions of enzyme (2 nmol of subunit/ml) in acetate buffer pH 6 were treated with less than stoichiometric amounts of permanganate (0), periodate (O), or hexachloroplatinate (A). Ten minutes after each addition a sample of enzyme was assayed for residual enzymatic activity.

manganate; in this case the formation of disulfide bridge or sulfinate groups appears to be excluded (32, 38). Identification of the Amino Acid(s) Involved in the Inactivation by Phosphate Analogs The enzyme, treated with several inactivating agents until a 90% inactivation was obtained, was examined by titration of SH groups according to Ellman (2) and by amino acid analysis after acid hydrolysis. Titration of the SH groups indicated the presence of an average of 1.8 SH groups per subunit, out of the 4 originally present. Amino acid analyses indicated that there

DEHYDROGENASE

521

was no difference in the content of any amino acid residue between the inactivated and untreated enzyme, except that the enzyme inactivated by permanganate contained 1.5 mol of cysteic acid per enzyme subunit mole. These results indicate that complete inactivation is correlated with the oxidation of 1.5 cysteine residues to cysteic acid (in the case of permanganate) or with the oxidation of two adjacent SH groups to form a disultide bridge (in the case of periodate or hexachloroplatinate). Assuming the permanganate to be reduced to manganate (31) the periodate to iodate and the hexachloroplatinate to tetrachloroplatinate, the titration (Fig. 9) and the oxidation to cysteic acid data account for the 75% of the permanganate added and the formation of the disulhde bridge accounts .for 66% and lOO%, respectively, of the periodate and the hexachloroplatinate added. On the basis of the topographical specificity of the affinity labelling, it can be postulated that in close proximity to the phosphate binding site there are two SH groups that can form a disulfide bond. The presence of adjacent SH groups has already been detected using a different approach (30). Other tetrahedral anions, such as chromate and perchlorate, also inactivate the enzyme, but the concentrations required are higher than that with permanganate and periodate. This appears to be the first report of the use of periodate and hexachloroplatinate as phosphate analogs, as enzyme inhibitor and for affinity labelling. Platinum derivatives have been used for x-ray analyses. If the specificity of hexachloroplatinate for phosphate binding sites is demonstrated also for other enzymes acting on phosphorylated compounds, this new reagent could be used for the preparation of heavy atom derivatives of these enzymes, having the advantage, over other platinum derivatives, that its binding site is easily localized as the phosphate binding site. The Presence of a Lysine Residue in the Vicinity of the Phosphate Binding Site In 1966 simultaneously

and independ-

522

RIPPA

ently in this (39-41) and in another (42) laboratory it was discovered that PLP can be used as an enzyme inhibitor specific for lysine residues. The fact that the pyridoxal is much less effective than PLP indicated a role for the phosphate group in the binding of the inhibitor to the enzyme. The PLP can be considered a bifunctional reagent able to bridge the e-amino group of a lysine residue to an amino acid residue involved in the binding of phosphate (or other) anion. Since this paper is concerned with the phosphate binding site of 6-phosphogluconate, we reinvestigated the inhibition by PLP and found that, as expected, phosphate and other tetrahedral anions protect against the PLP inhibition (Fig. 10). Repeating the kinetic analysis used for the other inhibitors, it appears that the rate of inhibition by PLP is a function of the concentration of the inhibitor, that the inhibition follows a saturation kinetics (Fig. ll), and that the Kinact of PLP is 0.38 mM. It has been reported that PLP, acting as enzyme inhibitor, binds to the enzyme first as a noncovalent complex which then forms a covalent complex (43-45) and thus can be used for affinity labeling. In the light of the experiments reported here, it may be postulated that the e-amino group of the lysine

ET AL.

FIG. 11. Determination of the Kin.,, of PLP. A solution of enzyme (2 nmol of subunit/ml) in TEA buffer, pH 7.5, was treated with PLP at different concentrations and the inactivation half time r at each concentration was calculated.

residue involved in the inactivation by PLP is not far from the phosphate binding site.

A Histidine Residue Near the Phosphate Binding Site We have also reported (46) that PLP can be used as an active site specific photosensitizer (47). We found that the photo-oxidation of the enzyme-PLP complex causes the inactivation of the enzyme, correlated to the specific oxidation of a single histidine residue per enzyme subunit. On the basis of the site specificity of photoinactivation and of the evidence that PLP bridges the amino acid(s) involved in the binding of the phosphate to the e-amino group of a lysine residue (both at the active site of the enzyme) it may be postulated that the imidazole group of the histidine residue destroyed by the photoinactivation is also located near the phosphate binding site and the lysine residue in the three-dimensional structure of the active site of the enzyme. CONCLUSIONS

FIG. 10. Kinetics of the inactivation of enzyme by pyridoxal 5’-phosphate. A solution of enzyme (2 nmol of subunit/ml) in 50 mM TEA buffer, pH 7.5, was treated with PLP (final concentration 25 mM). Symbols: 0, no other addition; 0, in the presence of 6PG or sulfate; A, in the presence of NADP+; A, in the presence of phosphate. The concentrations of 6PG, NADP’, and of the anions are as in the legend of Fig. 2.

It may be concluded that the phosphate binding site (probably an arginine residue) at the active site of phosphogluconate dehydrogenase acts as an anchor for many reagents able to bind to, or react with, amino acid residues that are close to the phosphate binding site in the three-dimensional structure of the active site. Using appropriate phosphate analogs we have detected the presence of a pair of cysteine

ACTIVE

SITE

OF 6-PHOSPHOGLUCONATE

residues able to form a disulfide bridge and lysine and histidine residues at that site. A tyrosine residue, shown to be close to a cysteine residue at the active site (29), should also be located in this domain. REFERENCES 1. RIPPA, M., AND SIGNORINI, M. (1975) in Methods in Enzymology (W. A. Wood, ed.), Vol. 41B, pp. 237-240, Academic Press, New York. 2. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82,70-77. 3. RIPPA, M., GRAZI, E., AND PONTREMOLI, S. (1966) J. Biol. Chem. 241, 1632-1635. 4. PROCSAL, D., AND HOLTEN, D. (1972) Biochemistv 11, 1310-1314. 5. DYSON, J. E. D. AND D’ORAZIO, R. E. (1973) J. Biol. Chem. 248,5428-5435. 6. PEARSE,

B. M. F., AND ROSEMEYER,

M. A. (1974)

European J. Biochem. 42, 213-223. 7. LEVY, H. M., LEBER, P. D., AND RYAN, (1963) J. Biol. Chem. 238,3654-3659. 8. SCRUTTON,

M.

C., AND

UTTER,

M.

E. M.

F. (1965)

Biol. Chem. 240,3714-3723. 9. KJZECH, D. B., AND FARRANT, R. K. Biochim. Biophys. Acta 151,493-503.

J.

(1968)

10. HOLLENBERG, P. F., FLASHNER, M., AND COON, M. J. (1971) J. Biol. Chem. 246, 946-953. 11. MARKUS, F., SCHUSTER, S. M., AND LARDY, H. A. (1976) J. Biol. Chem. 251, 1775-1780. 12. ANDREO, C. S., AND VALLEJOS, R. H. (1977) FEBS

Lett. 78, 207-210. 13. HUANG,

W. Y., AND TANG,

J. (1972) J. Biol. Chem.

247,2704-2710. 14. LANGE, G. I,., RIORDAN, J. F., AND VALLEE, B. L. (1974) Biochemistry 13,4361-4370. 15. PAL, P. K., AND COLMAN, R. F. (1976) European J. Biochem. 68, 437-442. 16. EHRLICH, R. S., AND COLMAN, R. F. (1977) Biochemistry 16,3378-3383. 17. YANKEELOW, J. A. (1970) Biochemistry 9, 2433-2439. 18. RIORDAN, J. F. (1973) Biochemistry 12.3915-3923. 19. LANGE,

G. I,., RIORDAN,

J. F., AND VALLEE,

B. L.

(1974) Biochemistry

13, 2865-2871. 20. LOBB, R. R., STOKES, A. M., HILL, H. A. O., AND RIORDAN. J. F. (1975) FEBS Lett. 54, 70-72. 21. ARNONE, A., BIER, C. J., COTTON, F. A., DAY, V. W., HAZE:N, E. E., RICHARDSON, D. C., RICHARDSON, ,J. S., AND YONATH, A. (1971) J. Biol.

Chem. 246, 2302-2316. 22. DAEMEN, F. J. M., AND RIORDAN, Biochemistry 13, 2865-2871.

J. F. (1974)

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23. COTTON, F. A., HAZEN, E. E., DAY, V. W., LARSEN, S., NORMAN, J. G., WONG, S. T. K., AND JOHNSON, K. H. (1973) J. Amer. Chem. Sot. 95,

2367-2369. S. G., AND RIORDAN, J. F. (1975) Proc. Nat. Acad. Sci. USA 72, 2616-2630. 25. LOBB, R. R., STOKES, A. M., HILL, H. A. O., AND RIORDAN, J. F. (1976) European J. Biochem. 70, 517-522. 26. BORDERS, C. L., AND WILSON, B. A. (1976) Biothem. Biophys. Res. Commun. 73, 978-984. 27. LEVY, H. R., INGULLI, J., AND AFOLAYAN, A. (1977) J. Biol. Chem. 252, 3745-3751. 28. RIPPA, M., SIGNORINI, M., AND PONTREMOLI, S. (1967) European J. Biochem. 1, 170-178. 29. DALLOCCHIO, F., SIGNORINI, M., AND RIPPA, M. (1978) Arch. Biochem. Biophys. 185,57-60. 30. RIPPA, M., SIGNORINI, M., PERNICI, A., AND DALI,OCCHIO, F. (1978) Arch. Biochem. Biaphys. 186,406-410. 31. RIPPA, M., SIGNORINI, M., AND PONTREMOLI, S. (1971) Arch. Biochem. Biophys. 150, 503-510. 32. BENISECK, W. F. (1971) J. Biol. Chem. 246, 3151-3159. 33. ROBERTS, F. M., SWITZER, R. L., AND SCHUBERT, R. K. (1975) J. Biol. Chem. 250, 5364-5369. 34. LEE, Y. M., AND BENISECK, W. F. (1976) J. Biol. Chem. 251, 1553-1560. 36. LAIDLER, K. (1958) The Chemical Kinetics of Enzyme Action, p. 23, Oxford, London. 36. MELOCHE, M. P. (1967) Biochemistry 6, 2273-2280. 37. WOLD, F. (1977) in Methods in Enzymology (Jacoby, W. B., and Wilchek, M., eds., vol. 46, pp. 3-14, Academic Press, New York. 38. TRUNDLE, D., AND CUNNINGHAM, L. W. (1969) Biochemistry 8, 1919-1923. 39. RIPPA, M., SPANIO, L., AND PONTREMOLI, S. (1966) Boll. Sot. Ital. Biol. Sper. 42, 748-749. 24. POWERS,

40. SPANIO, L., RIPPA, M., AND PONTREMOLI, S. (1966) Boll. Sot. Ital. Biol. Sper. 42, 750-751. 41. RIPPA, M., SPANIO, L., AND PONTREMOLI, S. (1967) Arch. Biochem. Biophys. 118,48-57. 42. ANDERSON, B. M., ANDERSON, C. D., AND CHURCHICH, J. E. (1966) Biochemistry 5, 2893-2909. 43. PISZKIEWICZ, D., AND SMITH, E. L. (1971) Bio-

chemistry

10,4538-4543.

44. CHEN, S. S., AND ENGEL

P. C. (1974) Biochem. J. 147,351-358. 45. VILJOEN, C., VISSER, L., AND BOTES, P. D. (1977) Biochim. Biophys. Acta 483, 107-120. 46. RIPPA, M., AND PONTREMOLI, S. (1969) Arch. Biothem. Biophys. 133,112-118. 47. RIPPA,

M., PICCO, C., AND PONTREMOLI,

J. Biol. Chem. 245.4977-4981.

S. (1970)