Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin]

The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK'1 6.1 and pK'2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.     

Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.     

High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.     

Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.     

Hyper production of enzyme penicillin amidase by locally isolated thermo-tolerantBacillus sp. MARC-0103 from rice starch in cheese whey

The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.     

Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data

The active site of lysostaphin is shown to contain a residue of glutamic acid. As judged by a pK value of 9.2 (with pentaglycine bridges in peptidoglycan of staphylococci as a substrate), another ionogenic residue could be the epsilon-amino group of a lysine. However, the pH value near a negatively charged cell is supposed to be strongly shifted to acidity as compared to the pH of the solution volume. This shifts the enzyme pH dependence curve in solution to alkalinity. Therefore, the other group might be histidine, which is consistent with the X-ray crystallographic data. A similar shift is likely to occur for lysozyme in the case of Micrococcus lysodeikticus cells. Determination of pK of ionogenic groups in the active sites of alkaline enzymes responsible for lysis of negatively charged bacterial cells gives their apparent values because the "pericellular" and "voluminous" values of pH are not coincident.     

pH dependence of penicillin amidase enantioselectivity for charged substrates.

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2). The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis. The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8. The dependence was stronger for the S'(1)- than for the S(1)-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.     

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Study of pH-dependence of kinetic parameters of pyruvate kinase from bovine adrenal cortex

The effect of pH on the main kinetic parameters of pyruvate kinase function was studied. The maximal rate of the reaction as well as the values of Km for ADP and Ki for phenylalanine depend on pH and show a well-defined extremum at pH 6.8-7.0. Spectrofluorimetric titration of pyruvate kinase results in pH dependencies of changes in the fluorescence spectra parameters (e.g., quantum yield, half-width and position of the maximum). This enabled to determine the pH regions corresponding to changes in the state of tryptophan residues. Data from the enzyme inhibition by phenylalanine suggest that acidification of the medium leads to the decrease of the catalytic activity due to the protonation of the ionogenic group of the enzyme. Within the pH range of 7.0-8.0, the decrease of the pyruvate kinase activity is due to structural shifts in the enzyme molecule, as a result of which the steric complementariness of the enzyme active center with respect to the substrate (Mg.ADP) is impaired.     

Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of ampicillin enzymatic hydrolysis]

The equilibrium parameters of the hydrolysis of ampicillin catalysed by penicillin amidase were determined within the pH range of 4.5 to 5.5. The values of the ionization constants of the carboxy group of D-(-)-ALPHA-AMINOPHENYLACETIC ACID (PK1=1.80) and amino group of 6-aminopenicillanic acid (pK2=4.60) were estimated and pH-dependence of the effective free energy of ampicillin hydrolysis was calculated. It was shown that the thermodynamic optimum of ampicillin synthesis was at 3.20 (the value of the effective free energy under the experimental conditions was 3.27 kcal/mole). The value of the "true", pH-independent free energy of hydrolysis (deltasigma) of the amide bond in the ampicillin molecule was determined to be equal to 9.72 kcal/mole. The thermodynamic parameters of ampicillin and benzylpenicillin hydrolysis were compared. The amino group in the alpha-position of phenylacetic acid was shown to have a significant effect on the values of "true" free energy of hydrolysis of the penicillin amide bond and free ionization energy in the system.     

Acetylcholinesterase-catalyzed hydrolysis of an amide.

In this paper we report that acetylcholinesterase catalyzes hydrolysis of amides, an observation which had not been made previously. The amide used is an analog of acetylcholine, 2-acetoaminoethyltrimethylammonium iodide. The experiments were performed with an enzyme preparation obtained from electroplax of Electrophorus electricus. Inhibition of the enzyme by a specific organic phosphate inhibitor abolished both the esterase and the amidase activity of the enzyme. The effect of hydrogen ions between pH 5 and pH 10 on the steady-state kinetic parameters, Km and kcat, has been investigated. These parameters show essentially the same dependence on pH as is observed in catalytic hydrolysis of acetylcholine. k-cat is controlled by an ionizing group of the enzyme with an apparent pK of approximately 6.3, and reaches a pH-independent maximum value of 3.6 sec- minus 1 above pH 8. The value for Km of 1 mM at pH 7 and 25 degrees is about five times greater than that for catalytic hydrolysis of the ester at the same pH and temperature. Preliminary electrophysiological experiments indicate that the amide analog binds to the receptor less well, by several orders of magnitude, than acetylcholine does.     

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.     

The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. Recently, the three-dimensional structure of the enzyme from yeast has been determined [Pang et al., J. Mol. Biol. 317 (2002) 249-262]. While this structure sheds light on the binding of the cofactors and the reaction mechanism, the interactions between the substrates and the enzyme remain unclear. We have studied the pH dependence of kinetic parameters in order to obtain information about the chemical mechanism in the active site. Data are consistent with a mechanism in which substrate selectively catalyzed to the enzyme with an unprotonated base having a pK of 6.48, and a protonated group having a pK of 8.25 for catalysis. The temperature dependence of kinetic parameters was pH-dependent, and the enthalpies of ionization, DeltaH(ion), calculated from the slope of pK(1) and pK(2) are both pH-independent. The solvent perturbation of kinetic parameters was pH-dependent, and the pK(1) from the acidic side and the pK(2) from the basic side were shifted down 0.4 pH units and shifted up 0.6 units as water was replaced by 15% ethanol, respectively. The data are discussed in terms of the acid-base chemical mechanism.     

Stabilisation and immobilisation of penicillin amidase

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.     

Conformation of the active site of thiolsubtilisin: reaction with specific chloromethyl ketones and arylacryloylimidazoles.

The conformation of the active site of thiolsubtilisin, prepared from subtilisin by transformation of the active site Ser to Cys, was compared with that of subtilisin by kinetic and spectroscopic methods. Carbobenzyloxy-L-alanylglycyl-L-phenylalanine chloromethyl ketone inhibited thiolsubtilisin approximately 10(2) times faster than subtilisin; alkylation occurred at the sulfhydryl rather than the imidazolyl group of the active site. pH dependence of the inhibition is different from that of the reaction between a simple thiol with haloacetamide. Furthermore, several native chromophoric arylacryloyl-thiolsubtilisins and arylacryloyl-subtilisins showed similar red shifts when compared with their denatured forms. The rate of deacylation of arylacryloyl-thiolsubtilisins was faster than (or of the same order of magnitude as) the deacylation rate of the analogous arylacryloyl-subtilisins in 30% dioxane (v/v), pH 5--10. The deacylation rate--pH profiles of these arylacryloyl-thiolsubtilisins in 30% dioxane all give pK values of 7.7 which is identical with the pK in the deacylation of acyl-subtilisins. These facts strongly suggest that the active-site conformation remains intact on conversion from subtilisin to thiolsubtilisin. The low esterase and peptidase activities of thiolsubtilisin are most likely due to the relatively low basicity of -SH (compared with -OH).     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). I. 1H NMR studies.

The basic pancreatic trypsin inhibitor (BPTI) was investigated by high resolution 1H NMR techniques at 360 MHz. Observation of the amide proton resonances of the polypeptide backbone showed that the globular conformation of BPTI determined by X-ray studies in single crystals is maintained in aqueous solution over the temperature range from 4 degrees to 87 degrees. NMR studies over this temperature range of the aromatic amino acid residues of BPTI. i.e. 4 tyrosines and 4 phenylalanines, led to complete assignments of all the aromatic spin systems in the protein. From this, information was obtained on the rotational motions about the C beta--Cv bond axis of the aromatic rings in the globular form of PBTI. At 25 degrees, two tyrosine rings and one phenylalanine ring are rotating rapidly on the NMR time scale. For the other rings the transitions from slow to rapid rotational motions were investigated at variable temperatures and energy barriers for these intramolecular rate processes determined. The studies of the tyrosine resonances had been described in detail in a previous publication. The present paper describes the identification of the phenylalanine resonances and comments on some technical aspects which might be of quite general interest for the analysis of highly resolved 1H NMR spectra of proteins. Data for the tyrosines and the phenylalanines are compiled in three tables, i.e. the pK alpha-values for the tyrosines, the NMR parameters for all eight aromatics, and the parameters delta G not equal to, and, where available, delta H not equal to and delta S not equal to for the rotational motions of the rings.     

Protonation of the binuclear metal center within the active site of phosphotriesterase

Phosphotriesterase (PTE) is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphates, including pesticides and chemical warfare agents, at rates approaching the diffusion controlled limit. The catalytic mechanism of this enzyme features a bridging solvent molecule that is proposed to initiate nucleophilic attack at the phosphorus center of the substrate. X-band EPR spectroscopy is utilized to investigate the active site of Mn/Mn-substituted PTE. Simulation of the dominant EPR spectrum from the coupled binuclear center of Mn/Mn-PTE requires slightly rhombic zero-field splitting parameters. Assuming that the signal arises from the S = 2 manifold, an exchange coupling constant of J = -2.7 +/- 0.2 cm(-)(1) (H(ex) = -2JS(1) x S(2)) is calculated. A kinetic pK(a) of 7.1 +/- 0.1 associated with loss in activity at low pH indicates that a protonation event is responsible for inhibition of catalysis. Analysis of changes in the EPR spectrum as a function of pH provides a pK(a) of 7.3 +/- 0.1 that is assigned as the protonation of the hydroxyl bridge. From the comparison of kinetic and spectral pK(a) values, it is concluded that the loss of catalytic activity at acidic pH results from the protonation of the hydroxide that bridges the binuclear metal center.     

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.     

Pigeon liver diacetyl reductase. Effects of pH on the kinetic parameters of the reaction.

(1) The pH dependence of the kinetic parameters of the reaction catalyzed by pigeon liver diacetyl reductase (EC 1.1.1.5) was investigated in the pH range 5.1-8.6. (2) From the results obtained it is postulated that: (a), a group of pK around 7, active in the protonated form, participates in the interaction of the enzyme with NADH and NAD. (b), a second group with a pK of 8.4, active in the protonated form too, takes part in the binding of diacetyl to E-NADH. (c) A third group of pK about 4.7-5, active in the unprotonated form, is involved at least in the dissociation of the complex E-NAD and in the attachment of diacetyl to E-NADH.