scholarly journals Chemical synthesis and papain-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide

1985 ◽  
Vol 226 (2) ◽  
pp. 601-606 ◽  
Author(s):  
N E Mackenzie ◽  
J P G Malthouse ◽  
A I Scott
Keyword(s):  
Chemical Synthesis ◽  
Ph Dependence ◽  
Low Ph ◽  
Pka Values ◽  
Tetrahedral Intermediate ◽  
Ph Values ◽  
Km Value ◽  
Hydrolysis Of

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.

pH Effects in trypsin catalysis

1969 ◽  
Vol 47 (21) ◽  
pp. 4021-4029 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Complex Formation ◽  
Ph Dependence ◽  
Specific Rotation ◽  
Ph Effects ◽  
Ph Values ◽  
A Value ◽  
Available Information ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Covalent Complex

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

Hydrolysis of N-acetyl-p-benzoquinone imines: pH dependence of the partitioning of a tetrahedral intermediate

1989 ◽  
Vol 111 (12) ◽  
pp. 4447-4456 ◽  
Author(s):  
Michael Novak ◽  
Gayl A. Bonham ◽  
Julio J. Mulero ◽  
Maria Pelecanou ◽  
Joseph N. Zemis ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Tetrahedral Intermediate ◽  
Hydrolysis Of

ChemInform Abstract: Hydrolysis of N-Acetyl-p-benzoquinone Imines: pH Dependence of the Partitioning of a Tetrahedral Intermediate

ChemInform ◽  
1989 ◽  
Vol 20 (39) ◽  
Author(s):  
M. NOVAK ◽  
G. A. BONHAM ◽  
J. J. MULERO ◽  
M. PELECANOU ◽  
J. N. ZEMIS ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Tetrahedral Intermediate ◽  
Hydrolysis Of

scholarly journals pH-dependence of the phospholipid interaction of diphtheria-toxin fragments

1985 ◽  
Vol 231 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Montecucco ◽  
G Schiavo ◽  
M Tomasi
Keyword(s):  
Diphtheria Toxin ◽  
Ph Dependence ◽  
Low Ph ◽  
Membrane Insertion ◽  
Ph Values ◽  
Egg Lecithin ◽  
A Chain ◽  
Lipid Interaction

Photoreactive phospholipids have been used to probe the lipid interaction of diphtheria toxin. Low pH values induce the membrane insertion of both the binding and enzymic fragments of the toxin. The efficiency of this process is much higher with asolectin than with egg lecithin (phosphatidylcholine)/cholesterol liposomes. The low-pH-induced interaction of the toxin fragments with the membrane hydrocarbon phase is more evident for the enzymic A-chain than for the binding B-chain, and it is fully reversed by returning the pH to neutrality.

scholarly journals The catalytic activity of pig pepsin C towards small synthetic substrates

1979 ◽  
Vol 179 (1) ◽  
pp. 239-246 ◽  
Author(s):  
C A Auffret ◽  
A P Ryle
Keyword(s):  
Catalytic Activity ◽  
General Formula ◽  
Competitive Inhibition ◽  
Ph Dependence ◽  
Small Peptides ◽  
Pka Values ◽  
A Minor ◽  
Hydrolysis Of

A series of small peptides has been synthesized and used to investigate the activity of a minor pig pepsin, pepsin C (EC 3.4.23.3). The peptides had the general formula A-Leu-Val-His-B. B was either OMe, NH2 or OH. With B = NH2 hydrolysis (kcat./Km) at 37 degrees C and pH 2.07 increased as A was Ac-Ala, Ac-Tyr, Ac-Phe and Ac-Ala-Phe. The pH dependence of the hydrolysis of Ac-Phe-Leu-Val-His-NH2 indicated the apparent pKa values of two catalytically important groups on the enzyme as 1.42 and 4.88. Inhibition of the hydrolysis of the same peptide by Ac-Phe at pH 3.01 showed a form of mixed non-competitive inhibition. Hydrolysis of Ac-Tyr-Leu-Val-His-OMe and the corresponding amide showed non-classical kinetics, which are discussed in terms of a substrate-activating mechanism. The results are discussed with reference to observations made by other workers on pig pepsin A.

Comparative Study of p-nitrophenyl picolinate Hydrolysis Catalyzed by Bis(O,O’-di(2-phenylalkyl)dithiophosphate) nickel(II)

2012 ◽  
Vol 560-561 ◽  
pp. 305-308 ◽  
Author(s):  
Bing Ying Jiang ◽  
Fa Mei Feng ◽  
Min Wang ◽  
Ci Li ◽  
Jia Qing Xie
Keyword(s):  
Comparative Study ◽  
Metal Complexes ◽  
Low Ph ◽  
Nucleophilic Attack ◽  
High Ph ◽  
Ph Values ◽  
Catalytic Function ◽  
Hydrolysis Of ◽  
Pnpp Hydrolysis ◽  
Synergism Effect

The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by two nickel (II) complexes (bis(O,O’-di(2-phenylmethyl) dithiophosphate) nickel(II) (NiR1) and bis(O,O’-di(2-phenylethyl) dithiophosphate) nickel(II) (NiR2)) was investigated kinetically in this work. The results indicate that both metal complexes accelerate the hydrolysis of PNPP dramatically and the NiR1 exhibits higher catalytic function on PNPP hydrolysis in the buffered solution with relatively low pH values, while NiR2 shows slightly more efficacy on hydrolysis of PNPP in relatively high pH buffered solutions. This variance is ascribed to the synergism effect of space hindrance of the complexes and the nucleophilic attack of metal-hydroxy species generated by the complexes.

scholarly journals The pH-dependence of pepsin-catalysed reactions

1969 ◽  
Vol 113 (2) ◽  
pp. 353-362 ◽  
Author(s):  
A. J. Cornish-Bowden ◽  
J. R. Knowles
Keyword(s):  
Continuous Monitoring ◽  
Ph Dependence ◽  
Preceding Paper ◽  
Free Enzyme ◽  
Peptide Substrates ◽  
Pka Values ◽  
Hydrolysis Of

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pKa values of 1·1 and 3·5 in the plots of k0/Km versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pKa values of 1·0 and 4·7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pKa values of 1·0 and 4·7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pKa values of 3·5 for the dependence of k0/Km for these materials.

scholarly journals Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding

1976 ◽  
Vol 157 (2) ◽  
pp. 415-421 ◽  
Author(s):  
K Airas
Keyword(s):  
Binding Site ◽  
Phosphate Buffer ◽  
Substrate Binding ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Inhibitor Binding ◽  
Substrate Binding Site ◽  
Ph Values ◽  
Km Value ◽  
Rate Profile

The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The Km value of pantothenase to pantothenate depends on the buffer used, and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6mM in phosphate buffer at 20 degrees C; the corresponding Kapp.m value at pH7 is 15 mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km rofile. pK is 7.0 in phosphate buffer at 20 degrees C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were—14kJ/mol respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the K1 values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K1 being 0.36mM, and the corresponding Kapp.m about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.

The mutation of Thr315 to Asn of GH10 xylanase XynR increases the alkaliphily but decreases the alkaline resistance

2021 ◽  
Author(s):  
Kohei Kuwata ◽  
Manami Suzuki ◽  
Teisuke Takita ◽  
Rie Yatsunami ◽  
Satoshi Nakamura ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Culture Broth ◽  
Saturation Mutagenesis ◽  
Ph Values ◽  
Beechwood Xylan ◽  
Alkaline Resistance ◽  
Dependent Activity ◽  
A Site ◽  
Hydrolysis Of ◽  
Gh10 Xylanase

Abstract XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type XynR (WT) and S92E. The optimal pH values of T315N and S92E/T315N were 6.5–9.5 while those of WT and S92E were 6.5–8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH-dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0–8.5 for T315N and S92E/T315N, but 6.0–10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.

scholarly journals The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre

1999 ◽  
Vol 338 (1) ◽  
pp. 235-239 ◽  
Author(s):  
Manuel MORILLAS ◽  
Martin L. GOBLE ◽  
Richard VIRDEN
Keyword(s):  
Rate Constant ◽  
Conformational Transition ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Penicillin G ◽  
Pka Values ◽  
Energy Compensation ◽  
Guanidinium Group ◽  
Kinetics Of ◽  
Hydrolysis Of

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.

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