Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Evidence for proximal cysteine and lysine residues at or near the ative site of arginine kinase of <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou's method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5 -dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274.     

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (GlDH EC 1.1.1.6), a tetrameric NAD + specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5′-phosphate (PLP) and o -phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced GlDH indicated the specific modification of ? -amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD + and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of GlDH by the bifunctional reagent OPA, which reacts specifically with proximal ? -NH 2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD + was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of GlDH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site

Yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a homodimer, was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics over a wide range of the inhibitor concentration. The second-order-rate constant for the inactivation of hexokinase was estimated to be 45 M-1.s-1. Hexokinase was protected more by sugar substrates than by nucleoside triphosphates during inactivation by o-phthalaldehyde. Absorption spectrum (lambda max 338 nm), and fluorescence excitation (lambda max 363 nm) and emission (lambda max 403 nm) spectra of the hexokinase-o-phthalaldehyde adduct were consistent with the formation of an isoindole derivative. These results also suggest that sulfhydryl and epsilon-amino functions of the cysteine and lysine residues, respectively, participating in the isoindole formation are about 3 A apart in the native enzyme. About 2 mol of the isoindole per mol of hexokinase dimer were formed following complete loss of the phosphotransferase activity. Chemical modification of hexokinase by iodoacetamide in the presence of mannose resulted in the modification of six sulfhydryl groups per mol of hexokinase with retention of the phosphotransferase activity. Subsequent reaction of the iodoacetamide modified hexokinase with o-phthalaldehyde resulted in complete loss of the phosphotransferase activity with concomitant modification of the remaining two sulfhydryl groups of hexokinase. Chemical modification of hexokinase by iodoacetamide in the absence of mannose resulted in complete inactivation of the enzyme. The iodoacetamide inactivated hexokinase failed to react with o-phthalaldehyde as evidenced by the absence of a fluorescence emission maximum characteristic of the isoindole derivative. The holoenzyme failed to react with [5'-(p-fluorosulfonyl)benzoyl]adenosine. The dissociated hexokinase could be inactivated by [5'-(p-fluorosulfonyl)benzoyl]adenosine; the degree of inactivation paralleled the extent of reaction between o-phthalaldehyde and the nucleotide-analog modified enzyme. Thus, it is concluded that two cysteines and lysines at or near the active site of the hexokinase were involved in reaction with o-phthalaldehyde following complete loss of the phosphotransferase activity. An important finding of this investigation is that the lysines, involved in isoindole formation, located at or near the active site are probably buried.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde

A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.     

Fluorescent labelling of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

The thermophilic 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus was inhibited upon specific modification of the -SH group of cysteine residues by 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) at pH 7.0. By using 20-100-fold molar excess of NBD-CL the reaction occurs slowly at pH 7.0 as a first order process. Partial protection from inactivation was observed when the substrate 6-phosphogluconate or the coenzyme NADP was added to the reaction mixture. Complete inactivation was achieved upon modification of 1.9 of the six cysteine residues per mole of enzyme, which corresponds to nearly one residue per enzyme subunit. Circular dichroism measurements suggest that the gross structure of the protein molecule is practically unchanged upon reaction of the enzyme with NBD-Cl. Melting profile experiments revealed a single transition occurring at about 65 degrees C. Analogously, the profile of intensity of the fluorescence emission at 520 nm of the enzyme-bound S-NBD groups versus temperature indicated a midpoint of transition near 65 degrees C. Since this melting temperature corresponds closely to that observed with the native enzyme, these results would indicate that the molecular organizations of the native and modified enzyme are similar and stabilized by similar interactions within the polypeptide chain.     

Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.     

The chemical modification of the essential groups of beta-N-acetyl-D-glucosaminidase from Turbo cornutus Solander

The chemical modification of beta-N-acetyl-D-glucosaminidase (EC3.2.1.30) from Turbo cornutus Solander has been first studied. The results demonstrate that the sulfhydryl group of cysteine residues and the hydroxyl group of serine residues are not essential to the enzyme's function. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 278 nm and the fluorescence intensity quenching at 335 nm, indicating that tryptophan is essential residue to the enzyme. The modification of amino group of lysine residue by formaldehyde and trinitrobenzenesulfonic acid also inactivates the enzyme completely. The results show that lysine and tryptophan are probably situated in the active site of the enzyme. The modification of the imidazole residue and carboxyl group leads to inactivate incompletely, indicating they are not the composing groups of the enzyme active center, and they are essential for maintaining the enzyme's conformation which is necessary for the catalytic activity of the enzyme.     

Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling. Site and significance of a histidine residue.

A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I-isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group-specific reagents and by amino-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I-isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.     

o-Phthalaldehyde as a probe in the active site of phosphoenolpyruvate carboxylase

Modification of phosphoenolpyruvate carboxylase with o-phthalaldehyde (OPA) resulted in rapid and irreversible inactivation exhibiting biphasic reaction kinetics. The kinetic analysis and correlation of spectral changes with activity indicated that inactivation by OPA results from the modification of two lysine and two cysteine residues per subunit of the enzyme. PEP plus Mg2+ offered substantial protection against modification. Some of the effectors also gave appreciable protection against modification indicating that the residues may be located at or close to the active site. Thus, the results indicate formation of two isoindoles showing the proximity of the essential lysine and cysteine residues at the active site.     

Sequential inactivation of zeta-crystallin by o-phthalaldehyde

o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens zeta-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of zeta-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with zeta-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified zeta-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of zeta-crystallin.     

Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a ''half-of-the-sites'' mechanism.

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn²⁺ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn²⁺ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn²⁺, indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn²⁺ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.     

Chemical modification of lysine residues at active-site of human placental estradiol 17 beta-dehydrogenase

Estradiol 17 beta-dehydrogenase (EC 1.1.1.62.) activity was decreased by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent for modification of epsilon-amino moiety of lysine residues in a protein. The inactivation exhibited pseudo-first-order kinetics, and was protected by oxidyzed cofactors. Stoichiometric studies showed that the complete inactivation was caused by modification of one lysine residue per molecule of the enzyme. Differential modification with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), TNBS and dithiothreitol (DTT) indicated that the residues of lysine and cysteine were located at the active-site and played an essential role in the catalytic function of the estradiol 17 beta-dehydrogenase.     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

Study of the amino acid residue topography of the adenosine triphosphatase center of (Ca--Mg)-dependent sarcoplasmic reticulum adenosine triphosphatase by kinetic and spectral methods

The topography of HS- and NH2-groups and tryptophane residues in ATPase centre of (Ca--Mg)-ATPase on sarcoplasmic reticulum (SR) was investigated by kinetics, electron spectroscopy and spectrofluorimetry method. Both o-phthalaldehyde interacting with lysine or arginine residue or with end amino acid and fluorescein dimercuric acetate interaction with cysteine residue of HS-groups make (Ca--Mg)-ATPase both in SR and the pure enzyme completely inactive at molar ratio enzyme: inhibitor equal to 1 : 1. A 500 molar ATP surplus reduces drastically the enzyme inactivation rate by both inhibitors. The data supplied by the spectrofluorimetry and the induction-resonance theory were used to calculate the distances between nearest tryptophane residues and chromophore (o-FTC) generated by o-phthalaldehyde interaction with NH2-group the protein amino acid residue (17 A) and o-FTC and fluorescein dimercuric acetate (19 A) attached to enzyme HS-group. Because o-FTC is inside the protein pocket it is not accessible to J- ions up to 2.5 M KJ. However some tryptophane resudies and fluorescein dimercuric acetate attached to HS-group are near to the macromolecule surface. Lysine (or arginine residues) or end amino acid NH2-group and cysteine residues HS-group, and some tryptophane residues are at ATPase centre of (Ca--Mg)-ATPase from sarcoplasmic reticulum. Possible topography of the centre is discussed.