Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.     

Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.     

On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine.

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.     

Reversible dissociation of threonine deaminase in an ilvl mutant of Saccharomyces cerevisiae

Summary An impaired threonine deaminase resulting from a point mutation ilv1-6 in the locus corresponding to the structural gene coding for this enzyme in Saccharomyces cerevisiae is more susceptible to inactivation by aging than the corresponding wild type enzyme. However, this impaired activity can be fully recovered by addition of differently inactivated extracts. This reactivation can be achieved by the sole addition of the coenzyme pyridoxalphosphate (PLP). It is time and concentration dependent. All these effects are less pronounced with the wild type enzyme than with the mutant enzyme. It has been shown by sedimentation in glycerol gradients that inactivation of the mutant enzyme (MW 197000) is accompanied by dissociation into two protomers (MW 107000). Such a dissociation might be a clue to explain the numerous consequences of the mutation on the kinetic properties of the impaired enzyme as they might reveal a modified association between subunits and protomers.This work was supported by grants from the DGRST-France; the CEA-France and the FRMF.     

Turnover of ammonium-inducible glutamate dehydrogenase during induction and its rapid inactivation after removal of inducer from Chlorella sorokiniana cells.

When ammonia was removed from Chlorella sorokiniana cells, which contain an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), the activity of this enzyme decayed with a half-life of approximately 8 min. By use of rocket immunoelectrophoresis, indirect immunoprecipitation, and indirect immunoadsorption (coupled with pulse-chase experiments with 35S-labeled sulfate), the rapid initial loss in activity was shown to be due to enzyme inactivation rather than degradation of NADP-GDH antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained with anti-NADP-GDH immunoglobulin G showed that enzyme inactivation is accompanied by the conversion of enzyme subunits (Mr = 59,000) to a protein with a molecular weight of 118,000. Because this protein was stable during boiling and in the presence of sodium dodecyl sulfate and high concentrations of mercaptoethanol or dithiothreitol, it was tentatively assumed to be a covalently linked dimer of enzyme subunits. Pulse-chase experiments showed that total NADP-GDH antigen was subject to rapid degradation (t 1/2 = 88 min) in induced cells, and the same degradation rate was maintained after removal of ammonia from induced cells.     

Cooperative subunit interactions in C-type inactivation of K channels.

C-type inactivation of potassium channels is distinct from N-terminal mediated (N-type) inactivation and involves a closing of the outer mouth of the channel. We have investigated the role of the individual subunits of the tetrameric channel in the C-type inactivation conformational change by comparing the inactivation rates of channels constructed from different combinations of subunits. The relationship between the inactivation rate and the number of fast subunits is exponential, as would be predicted by a cooperative mechanism where the C-type conformational change involves all four subunits, and rules out a mechanism where a conformational change in any of the individual subunits is sufficient for inactivation. Subunit interactions in C-type inactivation are further supported by an interaction between separate mutations affecting C-type inactivation when in either the same or separate subunits.     

Modification of phenylalanyl-tRNA-synthetase from Escherichia coli MRE600 by adenosine-5'-trimetaphosphate

Modification of phenylalanyl-tRNA synthetase from E. coli MRE600 by adenosine-5'-trimetaphosphate, phosphorylating analog of ATP was shown to bring about the enzyme inactivation in the reactions of tRNA aminoacylation and ATP-[32P]pyrophosphate exchange. ATP when added in the reaction mixture protects the enzyme against inactivation in both reactions and decreases the level of covalent attachment of the analog. Phenylalanine has no protective effect. tRNA exhibits slight protective effect. Adenosine-5'-trimetaphosphate modifies both types (alpha and beta) of subunits of phenylalanyl-tRNA synthetase which is of alpha 2 beta 2 structure. ATP protects both types of the enzyme subunits against the covalent attachment of the analog. Disposition of the ATP-binding centers in the contact region of the nonequivalent subunits of the enzyme was proposed. The level of covalent attachment of the analog to the enzyme exceeds the number of the enzyme active sites that may be a consequence of the other nucleotide-binding center labeling.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Non-equivalency of SH groups essential for the activity of mitochondrial creatine kinase

The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.     

Differential sensitivities of the subunits of mammalian ribonucleotide reductase to proteases, sulfhydryl reagents, and heat

Ribonucleotide reductase catalyzes the rate-limiting step in the formation of 2'-deoxyribonucleoside 5'-triphosphates. It consists of two nonidentical protein subunits, the nonheme iron subunit, and the effector-binding subunit. It has previously been shown that these two components making up the active enzyme species are not coordinately synthesized or degraded. It was found that the effector-binding subunit was more sensitive to proteolysis by chymotrypsin, to heating at 55 degrees C, and to the sulfhydryl reagents, pCMB and NEM. The nonheme iron subunit was more sensitive to trypsin treatment. ATP and dATP protected the effector-binding subunit from proteolytic inactivation. Neither ATP nor CDP protected the effector-binding subunit from inactivation by the sulfhydryl reagents. These data indicate that the protein properties of the two subunits of mammalian ribonucleotide reductase are significantly different.     

Purification and properties of subunits of sterol ester hydrolase from rat pancreas.

A procedure has been developed for solubilizing subunits of sterol ester hydrolase (EC 3.1.1.13) from rat pancreas by treatment of a tissue homogenate with 0.5% ($\text{w}\text{v}$) digitonin. The crude, solubilized enzyme subunit was shown to have a molecular weight of approximately 70,000 by Sephadex G-200 gel filtration. This enzyme subunit has been purified 500-fold by a combination of ammonium sulfate fractionation, hydroxylapatite, and gel-filtration chromatography. The 70,000 molecular-weight subunits have been shown to aggregate in the presence of cholic acid or sodium taurocholate to a 400,000 molecular-weight form which is the active enzyme. Studies on binding of cholic acid to the subunit protein suggest that after the binding of one molecule of the bile acid, the subunit undergoes a conformational change(s) which makes additional binding sites for cholic acid available. Three types of differential inactivation studies (thermal, guanidine hydrochloride, and pH) indicated significantly greater stability of the active enzyme when compared to the subunits. These data are consistent with the tentative conclusion that a conformational change(s) accompanies the binding of the bile salt to the enzyme subunits, which results in their aggregation and enzyme activity. The theoretical and physiological significance of this interaction between the subunit protein and bile salt is discussed.     

myo-Inositol oxygenase from rat kidneys. I: Purification by affinity chromatography; physical and catalytic properties.

Using the technique of affinity chromatography on a myo-inositol-substituted Sepharose, the myo-inositol oxygenase from rat kidneys was purified to homogeneity. The active enzyme contains iron, most probably in its divalent form. Electrophoresis on polyacrylamide gel containing sodium dodecylsulphate causes the cleavage of the enzyme protein into apparently identical subunits with a molecular weight of approximately 17,000. The smallest active unit consists of 4 subunits, and is in a pH-dependent equilibium with species consisting of 8, 12, and 16 subunits, respectively, which all show the same specific enzyme activity. In the presence of oxygen the enzyme is highly unstable; at the early stages of inactivation it can be reactivated by reducing agents like NaBH4. Under anaerobic conditions or under the influence of Fe2-chelating agents, the enzyme is also inactivated; this inactivation is caused by the loss of iron and concomitant cleavage into the subunits. It can be reversed by incubation with FeSO4 in the presence of air. If myo-inositol and FeSO4 are present, the reactivation involves an oligomerization to the species with 16 subunits with the uptake of 8 gram-atoms of iron per mole of this species. The enzyme reaction follows Michaelis-Menten kinetics; the Michaelis constants are 4.5 x 10(-2)M for myo-inositol and 9.5 x 10(-6)M for oxygen.     

Reversible dissociation of malate dehydrogenase from plants]

It was demonstrated that 0.2 M citric acid (pH 2.5) inactivates highly-purified malate dehydrogenase from tea leaves; the degree of inactivation depends on temperature and time of incubation. The enzyme activity is restored by certain inorganic salts, the degree of reactivation being dependent on pH, ionic strengths of salts and duration of enzyme incubation with both inactivating and reactivating agents. Urea and guanidine hydrochloride also have a reversibly inactivating effect on the enzyme. The degree of inactivation depends on their concentration and incubation time. In the latter case reactivation of enzyme is achieved by dialysis or 20-40-fold dilution of the enzyme preparation. A kinetic study demonstrated that inactivation of enzyme by the above-mentioned agents is due to the enzyme dissociation into 4 catalytically inactive subunits with molecular weights of 17 500 +/- 1000, which under certain conditions are capable of reassociating into an active molecule of enzyme with completely restored native conformation.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid.

A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase.     

Hydrodynamic properties of porin isolated from outer membranes of rat liver mitochondria

The reaction of gamma-glutamyltranspeptidase with phenobarbital or with thiobarbituric acid resulted in a irreversible loss of its enzymatic activity. The inactivation followed pseudo-first-order kinetics. Half-maximal velocity of inactivation (Ki) at 37 degrees C in the presence of phenobarbital or thiobarbituric acid was calculated to be 43 mM and 20 mM, respectively. The inactivation of the enzyme activity by both these inhibitors was prevented by serine borate, a known competitive inhibitor, and by the substrate, reduced glutathione, suggesting an active-site-directed nature of the these inhibitors. Maleate provided slight protection against inactivation by thiobarbituric acid. Complete inactivation of the enzyme with tritium-labeled phenobarbital resulted in a stoichiometric incorporation of radioactivity into the enzyme protein. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis of tritium-labeled phenobarbital-enzyme complex, nearly all the radioactivity was found to be associated with the small subunit (Mr = 22 000) of the enzyme, indicating that the catalytic component of the enzyme is on the small subunits.     

Involvement of tryptophans at the catalytic and subunit-binding domains of transcarboxylase

Transcarboxylase from Propionibacterium shermanii is a multisubunit enzyme. It consists of one central hexameric subunit to which six outer dimeric subunits are attached through twelve biotinyl subunits. Both the central and the outer subunits are multi-tryptophan (Trp) proteins, and each contains 5 Trps per monomer. The roles of the Trps during catalysis and assembly of the enzyme have been studied by using N-bromosuccinimide (NBS) oxidation as a probe. Modification of approximately 10 Trps of the total 90 Trps of the intact enzyme results in loss of activity. Both the substrates, viz., methylmalonyl-CoA and pyruvate, afford protection (approximately 50%) against inactivation caused by NBS. Analyses of tryptic peptide maps and intrinsic fluorescence studies have indicated that modification of 10 Trps of the whole enzyme does not cause extensive conformational changes. Therefore, the Trps appear to be essential for catalytic activity. NBS modification of the individual subunits at pH 6.5 has demonstrated differential reactivity of their Trps. Modification of the exposed/reactive Trps of either one of the subunits significantly affects the subunit assembly with the complementary unmodified subunits to form active enzyme. It is proposed that Trps are involved at the subunit-binding domains of either the central or the outer subunit of transcarboxylase, in addition to those critical for catalysis.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

S-adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine

Homogeneous S-adenosylhomocysteinase (AdoHcyase) from rat liver is a tetrameric enzyme that contains four molecules of tightly bound NAD per mole of enzyme. We report here that incubation of the rat liver enzyme with ATP, Mg2+, and KCl leads to conversion of the active enzyme to an inactive form with release of all enzyme-bound NAD which can be recovered quantitatively by gel filtration. At various concentrations of ATP, the release of NAD corresponds closely with the degree of inactivation, suggesting that the four subunits are equivalent. Hydrolysis of ATP is not required for the inactivation process since nonhydrolyzable ATP analogues can replace ATP in the inactivation process. The ATP-dependent inactivation is fully reversible upon incubation of the inactivated enzyme with NAD. The ATP-dependent inactivation of the enzyme appears to be analogues to the cAMP-dependent inactivation of the enzyme from Dictyostelium discoideum described earlier by Hohman et al. (1985) [Hohman, R. J., Guitton, M. C., & Veron, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4578-4581; Hohman, R. J., Veron, M., & Guitton, M. C. (1985) Curr. Top. Cell. Regul. 26, 233-245] but differs from the irreversible inactivation studied earlier by Abeles et al. (1982) [Abeles, R. H., Fish, S., & Lapinskas, B. (1982) Biochemistry 21, 5557-5562]. These authors have ascribed the time-dependent inactivation that results from incubation of the enzyme with 2'-deoxyadenosine at the C-3' and concluded that AdoHcyase "probably consists of two nonequivalent pairs of subunits".(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.