Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation. The results show that there are two first-order inactivation constants which depend on pH and that the transition between them has a pK of 5.9. As the constants of ultrasonic inactivation are very dependent on the accessibility of the residues it is concluded that the conformational change modifies the accessibility of the active site.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

Investigation of the active site of the extracellular beta-D-xylosidase from Aspergillus carbonarius

The catalytic amino acid residues of the extracellular beta-D-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl beta-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group--presumably a histidine residue--took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification V(max) decreased to 10% of that of the unmodified enzyme and K(m) remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-beta-d-xylopyranosylamine for beta-D-xylosidase. The A. carbonarius beta-D-xylosidase was irreversible inactivated by N-bromoacetyl-beta-D-xylopyranosylamine. The competitive inhibitor beta-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Role of the reactive cysteine residue in restriction endonuclease Cfr9I.

Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding 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.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity. During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.     

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.     

Coenzyme B12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.     

Inactivation of leucine aminotransferase with diethylpyrocarbonate and rose bengal: evidence for an active site histidine residue

Modification of leucine aminotransferase by diethylpyrocarbonate or rose bengal-sensitized photo-oxidation caused rapid inactivation of the enzyme. The inactivation of leucine aminotransferase depended on the concentration of the reagent, the time of incubation and exhibited pseudo-first order kinetics. Rose bengal-sensitized photo-oxidation was maximum at pH 6.5 and 9. Substrates leucine and alpha-ketoglutarate protected the enzyme against inactivation by these reagents, thus suggesting participation of histidine residue at the substrate binding site.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Stimulation of galactosyltransferase in liver microsomes by lysolecithin

Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis.     

Reaction of alpha-mannosidase from Phaseolus vulgaris with group-specific reagents. Essential carboxyl groups.

When the pKm of alpha-mannosidase was determined at different pH values, the results indicated that ionizable groups with pK values of approx. 3.8 and 5.7 could be essential. Modification with carbodiimide or Woodward's Reagent K abolished the enzyme activity. The substrate analogue, alpha-methyl-D-mannoside, protected the enzyme against inactivation. Incorporation of a 14C-labeled nucleophile reagent in the presence or absence of the analogue suggested that 2--4 carboxyl groups were protected. Exchange studies indicated that the essential Zn2+ could be bound to such groups. There was no indication that hydroxyl groups, sulphydryl groups, guanidino groups or amino groups take part in the catalytic activity.