Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

Functional site of human placental estradiol 17 beta-dehydrogenase involves tyrosine residues, as evidenced by reaction to p-nitrobenzenesulfonyl fluoride

Native estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from human placenta was inactivated in time dependent manner by p-nitrobenzenesulfonyl fluoride (NBSF), which is a reagent for chemical modification of tyrosine. The sulfhydryl-blocked enzyme by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was also reacted with NBSF more slowly in pseudo-first-order kinetics. After the sequential treatments with DTNB, NBSF and dithiothreitol (DTT), the enzyme in which tyrosine residues alone were modified was isolated, and its activity was decreased. These results suggest that tyrosyl residues of the estradiol 17 beta-dehydrogenase from human placenta are located at or near its catalytic site, and play a functional role in the enzyme reaction.     

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.     

Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.     

Affinity labeling of the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta by 5'-p-fluorosulfonylbenzoyl adenosine

An analogue of adenosine nucleotide, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSB-Ado), appears to interact irreversibly with the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta. This conclusion is based on the following observations: (1) The estradiol 17 beta-dehydrogenase is inhibited by 5'-FSB-Ado. When NAD+ is the variable component in the presence of saturated amount of steroid, the type of the inhibition is competitive in nature. When the steroid is the variable component, mode of the inhibition becomes non-competitive. The results suggest reversible binding of 5'-FSB-Ado to the cofactor-binding site of the dehydrogenase. (2) 5'-FSB-Ado inactivates irreversibly the estradiol 17 beta-dehydrogenase in time- and concentration-dependent manners, following pseudo-first-order kinetics. But, no inactivation is observed in the presence of p-fluorosulfonylbenzoic acid, suggesting that adenosine moiety of 5'-FSB-Ado is essential for the affinity labeling of estradiol 17 beta-dehydrogenase. (3) NADP+ protects completely estradiol 17 beta-dehydrogenase from the inactivation of 5'-FSB-Ado, whereas NAD(H) is partially protective against the inactivation, suggesting that phosphate moiety at 2'-position of NADP+ disturbs the covalent binding of 5'-FSB-Ado at or near the cofactor-binding site of the enzyme. (4) 2',5'-ADP shows the significant protection against the inactivation by 5'-FSB-Ado, but less effect is observed in the presence of nicotinamide mononucleotides. These results suggest that 5'-FSB-Ado is an affinity ligand for binding-site of adenosine nucleotide moiety of the cofactor.     

Chemical modification of lysine residues in tyrosyl-tRNA-synthetase from cattle liver using pyridoxal-5'-phosphate]

Chemical modification of lysine residues of eukaryotic tyrosyl-tRNA synthetase was studied. It was shown that only four out of 22 lysine residues per enzyme dimer could be modified with pyridoxal-5'-phosphate. This modification led to the inactivation of tRNATyr aminoacylation by more than 90% but did not practically affect the rate of ATP-[32P]pyrophosphate exchange. Low molecular weight substrates (ATP, ATP-tyrosine) weakly protected the enzyme from inactivation, whereas tRNATyr afforded a much more effective protection. It was supposed that lysine residues of tyrosyl-tRNA synthetase can be involved in the interaction with tRNATyr.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

Affinity labeling of cofactor-binding region of human placental estradiol 17 beta-dehydrogenase by periodate-oxidized NADP+ (o-NADP+)

Periodate-oxidized NADP+ (o-NADP+), an analogue of the cofactors, is a reversible inhibitor of estradiol 17 beta-dehydrogenase in human placenta. Mode of the inhibition by o-NADP+ appeared to be competitive type (Ki = 0.84 microM) against NAD+ and non-competitive type (Ki = 1.13 microM) against estradiol, respectively. Treatment of the estradiol 17 beta-dehydrogenase with o-NADP+ resulted in time-dependent loss of the enzyme activity. The inactivation exhibited pseudo-first order kinetics (t1/2 = 15 min) and was protected by NAD+ and NADP+. On the other hand, periodate-oxidized ATP inactivated slightly the estradiol 17 beta-dehydrogenase. These results indicate that the residue(s) of lysines is located near the cofactor-binding region of estradiol 17 beta-dehydrogenase of human placenta.     

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.     

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.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Chemical modification of lysine residues in bacterial formate dehydrogenase

Specific modification of 4.4 lysine residues per molecule of formate dehydrogenase, from the methylotrophic bacterium Achromobacter parvulus I by pyridoxal, results in complete inactivation of the enzyme. The concentration effect of the modifying agent and substrates on the inactivation of formate dehydrogenase has been studied. Coenzymes do not protect the enzyme from inactivation. Complete maintenance of enzyme activity was achieved in the presence of saturating concentrations of the formate and upon formation of the ternary complex, enzyme-NAD-azide. Formate specifically protects two lysine residues per dimer molecule of the enzyme from modification. The presence of one essential lysine residue in the substrate-binding region of the enzyme active site is assumed.     

Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase).

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Studies on determination of active site amino acid residues in glyoxylate synthetase from potato tuber chloroplasts

A homogeneous preparation of glyoxylate synthetase from greening potato tubers was used to study the functional role of disulphide groups, lysine and tryptophan residues in enzyme catalysis. The formation of a thioisoindole derivative was demonstrated by spectral analysis of the reduced and o-phthalaldehyde-treated enzymes. o-Phthalaldehyde modification resulted in about a 25 % loss of tryptophan emission at 336 nm and the appearance of a 410-nm emission peak characteristic of a thioisoindole. Ferrous iron was capable of generating thiol groups and addition of substrate resulted in a faster disappearance of these thiols. The optimal time for maximum glyoxylate synthesis by glyoxylate synthetase paralleled the disappearance of these thiols. Involvement of lysine and tryptophan residues in the enzyme reaction was demonstrated by the inhibition of activity by pyridoxal 5′-phosphate and dimethyl(2-hydroxy 5-nitrobenzyl) sulphonium bromide (DMHNB), respectively. Pyridoxal phosphate strongly and reversibly inhibited glyoxylate synthetase, and substrate and metal ion provided significant protection against inhibition. The results suggest that the lysine residue may be at or near the active binding site. The lysyl residue formed a Schiff base with pyridoxal phosphate which was stabilised by NaBH₄. Glyoxylate synthetase was also irreversibly inactivated by a tryptophan selective reagent, DMHNB, while substrate provided substantial protection against inactivation. Kinetic analysis and correlation of the spectral data at 410 nm indicated that complete inactivation by DMHNB resulted from the modification of 5 tryptophan residues/subunit, of which one was essential for activity. The available evidence suggests a possible concerted action of enzyme disulphides, ferrous iron, lysine and aromatic amino acid residues in the synthesis of glyoxylate by this enzyme.     

Mutants of immunotoxin anti-Tac(dsFv)-PE38 with variable number of lysine residues as candidates for site-specific chemical modification. 1. Properties of mutant molecules

Chemical modification of proteins with substances such as poly(ethylene glycol) can add useful properties to proteins. Currently PEGylation is done in a random manner utilizing amino residues dispersed throughout a protein. For proteins such as immunotoxins, which have several different functional domains, random modification leads to inactivation. To determine if we could produce an immunotoxin with a diminished number of lysine residues so that chemical modification could be restricted to certain regions of the protein, we chose the recombinant immunotoxin anti-Tac(dsFv)-PE38 that has 13 lysine residues in the Fv portion and 3 in the toxin. We prepared a series of mutants with 0-12 lysines in the Fv and 0 or 3 in the toxin. Almost all of these molecules retain full biological activity. Our data indicate that replacement of lysine residues can be achieve without loss of biological potency. These molecules are a useful starting point to carry out site-specific PEGylation experiments.     

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.     

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.     

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.     

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.