Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

PAR对氨基酰化酶不可逆抑制动力学研究

本文将邹氏的在酶的活性修饰剂存在下的底物反应动力学理论应用于氨基酰化酶被金属螯合剂PAR脱锌而失活的动力学研究。通过对不同浓度的PAR存在下底物反应过程和含有PAR的不同浓度的底物中酶促反应的分析,讨论了PAR对氨基酰化酶的脱锌机制。这一过程很可能按如下机制进行:首先,PAR与酶分子活性部位的锌结合,形成一复合物,这一步是较快的反应,然后发生一个可逆的构象变化,最后是不可逆的去锌步骤。锌的存在显然稳定了酶活性部位的构象,而这正是酶活性所必需的。     

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.     

Contribution to Catalysis and to Stability of the Essential Cysteine Residue at the Active Site of d-Glucosaminate Dehydratase from Pseudomonas fluorescens

Chemical modification of purified d-glucosaminate dehydratase (GADH) apoenzyme by N-ethyl-maleimide (NEM) and by 7-chloro-4-aminobenzo-2-oxa-1,3-diazole (NBDC1) resulted in the time- and concentration-dependent inactivation of the enzyme in each case. The inactivation followed pseudo-first-order kinetics and a double-logarithmic plot of the observed pseudo-first-order rate constant against reagent concentration proved evidence for an approximately first-order reaction, suggesting that the modification of a single cysteine residue per mole of enzyme resulted in inactivation. Amino acid analysis of the NEM-inactivated enzyme showed that three moles of cysteine residues among six moles per mole of subunit were modified under these conditions, therefore one of the three cysteine residues modified by NEM may be essential for activity. Pyridoxal 5′-phosphate (PLP) and D-glucosaminate (GlcNA) protected the enzyme against inactivation by NEM and NBDCI. The apoenzyme was inactivated by EDTA and activity of enzyme was restored by incubation with Mn²⁺ in the presence of PLP. Incubation of the EDTA-treated enzyme with NEM inhibited the restoration of activity. These results suggest that one of the cysteine residues of GADH may be chelated to a Mn²⁺ at or near the active site of GADH, contributing to formation of the active enzyme.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

An essential arginine residue at the substrate binding site of 4-hydroxyisophthalate hydroxylase

4-Hydroxyisophthalate hydroxylase was inactivated by treatment with phenylglyoxal by a process obeying pseudo-first order kinetics indicating the presence of an essential arginine located presumably in the active site. Addition of saturating amounts of 4-hydroxyisophthalate during the treatment resulted in complete protection of the enzyme from the inactivation, but addition of NADPH was totally ineffective. Analysis of the effect of various substrate analogs on the protection of the enzyme showed that carboxyl and hydroxyl groups at para positions on the aromatic ring are essential for substrate binding to the active site. It was also observed that analogs which protect the enzyme against phenylglyoxal inactivation are themselves effective inhibitors of the enzyme activity.     

Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics

The kinetics of the inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid are described. Loss of beta-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm, and by loss of tritium from 6 alpha-[3H]-6 beta-bromopenicillanic acid. It is shown that all of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6 beta-bromopenicillanic acid reacts directly with the beta-lactamase active site. It is proposed that this inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6 beta-bromopenicillanic acid is thus a suicide substrate.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.     

Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase

Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

Presence of essential histidine residues in nadp-malic enzyme from maize

Modification of maize leaf NADP-malic enzyme by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation followed pseudo-first-order reaction kinetics. The inactivation of the enzyme showed saturation kinetics with a half inactivation time, at saturating DEP, equal to 0.15 min and K_DEP = 20 mM. The rate of inactivation was faster at 25° as compared to 0° (t_0.5 0.75 min at 25° as against 5.6 min at 4° at 5 mM DEP). The enzyme was partially protected against DEP inactivation by NADP and complete protection was seen in the presence of NADP + Mg²⁺ + malate or its analogues, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.25 M NH₂OH and almost complete recovery of the enzyme activity was also observed. The results suggest that DEP modifies 3.0 residues per subunit and of these at least two residue per subunit can be modified without loss of activity in the presence of substrate. Modification of about one histidine residue is correlated with the loss of enzyme activity.     

Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase

Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.     

Inactivation of intestinal alkaline phosphatase by inositol hexaphosphate-Cu (II) coordinate complexes

Alkaline phosphatase (APase) was greater than 99% inactivated upon incubation with myo-inositol hexakisphosphate (IHP) and Cu(II) ions. In the absence of Cu(II), IHP did not inactivate the enzyme. Likewise, cupric ions alone did not produce inactivation. Reactions of APase with IHP plus Cu(II) were competitively inhibited by zinc ions. In contrast to the marked effect of (IHP-Cu) chelate complexes on APase activity, the complexes of IHP with either Zn(II) or Mn(II) had no discernable effect. Both the extent and the rate of activity loss were dependent on the combined IHP and Cu(II) concentration. At an IHP to Cu(II) ratio of 11.6, the extent of inactivation was approximately proportional to the Cu(II) concentration with maximal inactivation attained above 10 microM. Under the same conditions, a nonlinear relation (saturation kinetics) was observed between the pseudo first-order rate constants for the reaction and the IHP and Cu(II) concentration. On the basis of adherence of the data to a mechanism involving an intermediate whose concentration was rate determining, it was suggested that a ternary complexes composed of the apoprotein, the catalytic site zinc ions, and one or more specific IHP-Cu(II) complex [( IHP-Cu]*) may be the first step along the reaction coordinate. Relevant to this possibility which assumes active site interaction is the fact that both IHP alone and (IHP-Cu) complexes are good competitive inhibitors of p-nitrophenyl phosphate hydrolysis under the same solution conditions wherein APase inactivation occurs in the absence of substrate. Rates of enzyme inactivation are decreased with an increase in pH from 6.5 to 8.0. They are also dependent upon buffer type and concentration, apparently related to their association constants for cupric ion binding. Over and above such specific effects, rates of inactivation are also reduced with an increase in ionic strength. Depending on the ratio and concentrations of IHP and Cu(II) used in the reaction with APase, subsequent exposure to EDTA followed by assay in the presence of Zn(II) gave recoveries of activity ranging from 60% to 100%. Both the prior inactivated enzyme (containing IHP and cupric ions) in the presence of EDTA and the native APase upon simultaneous exposure to IHP, Cu(II), and EDTA were slowly and irreversibly inactivated. Correction for this effect gave reconstitution of activity of the (IHP-Cu)-inactivated APase by Zn(II) addition equivalent to that which could be obtained by EDTA-treatment of the native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Substrate specificity and binding loci for inhibitors in an aminopeptidase purified from the plasma membrane of midgut cells of an insect (Rhynchosciara americana) larva

Plasma membrane-bound aminopeptidases (EC 3.4.11.2) are found in the midgut cells from Rhynchosciara americana larvae, and are recovered in soluble form after papain treatment. The major papain-released aminopeptidase (Mr 207,000 and pI 7.8) was shown to be a true aminopeptidase with a broad specificity toward aminoacyl-beta-naphthylamides and to be more active on tetra and tripeptides than on dipeptides. The purified aminopeptidase is inactivated by EDTA according to a kinetics which is half order in relation to EDTA. Leucine hydroxamate (Ki 27 microM) and hydroxylamine (Ki 5.4 mM) completely protect the enzyme from inactivation by EDTA, whereas isoamyl alcohol (Ki 62 mM) increases the inactivation rate. There are 2.3 binding sites in the enzyme for phenanthroline, which makes the binding of the substrate in the enzyme difficult, changes the enzyme-substrate into a more productive complex, and increases the inactivation rate of the enzyme by EDTA by 87-fold. The data support the proposal that the enzyme has a metal ion which is catalytically active and that the enzyme displays two subsites in its active center: a hydrophobic subsite, to which isoamyl alcohol binds exposing the metal ion, and a polar subsite, to which hydroxylamine binds.     

Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine

Rat liver glycine methyltransferase is inactivated by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) in a pseudo-first order fashion at pH 7.5. The addition of dithiothreitol (20 mM) to the reaction mixture results in partial restoration of enzyme activity. A semilog plot of residual activity after dithiothreitol reactivation versus time is also linear, indicating that at least two essential residues are present on the enzyme and the modification of either of which causes total loss of activity. The inactivation is accompanied by incorporation of the radiolabel from adenine-labeled FSBA, but the amount of radioactivity fixed is not altered upon treatment with dithiothreitol. From this fact and the stoichiometry between the loss of dithiothreitol-sensitive activity and the number of sulfhydryl groups disappeared, it is suggested that the dithiothreitol-sensitive inactivation is the consequence of the FSBA-mediated formation of a disulfide between two sulfhydryl groups in close proximity. Although 4 mol of reagent are covalently bound per enzyme subunit, the kinetics of modification and inactivation show that the reaction at 1 residue, which is identified as tyrosine, is responsible for the dithiothreitol-insensitive inactivation. The substrate S-adenosylmethionine provides complete protection against both types of inactivation, but the dithiothreitol-insensitive inactivation is protected much more effectively with a Kd value comparable to the Km value. This suggests that the tyrosine is located at or near the active site of the methyltransferase.