Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion

The acylation and deacylation stages of the hydrolysis of N-acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential pKa values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp161 is the source of a key electrostatic modulator (pKa 5.0±0.1) in actinidin, analogous to Asp158 in papain, whose influence is not detected kinetically; it is always in the ‘on’ state because of its low pKa value (2.8±0.06).

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The pH Dependence of the Steady State Kinetic Parameters for Staphylococcal Nuclease-catalyzed Hydrolysis of Deoxythymidine-3′-phosphate-5′-p-nitrophenylphosphate in H2O and D2O

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43731-9 ◽

1973 ◽

Vol 248 (13) ◽

pp. 4769-4774

Author(s):

Ben M. Dunn ◽

Carlo DiBello ◽

Christian B. Anfinsen

Keyword(s):

Steady State ◽

Kinetic Parameters ◽

Ph Dependence ◽

Staphylococcal Nuclease ◽

Steady State Kinetic ◽

Hydrolysis Of ◽

State Kinetic

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Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II

Biochemical Journal ◽

10.1042/bj2230649 ◽

1984 ◽

Vol 223 (3) ◽

pp. 649-657 ◽

Cited By ~ 7

Author(s):

K Brocklehurst ◽

P R Carey ◽

H H Lee ◽

E Salih ◽

A C Storer

Keyword(s):

Steady State ◽

Kinetic Study ◽

Close Contact ◽

Cysteine Proteinase ◽

Resonance Raman ◽

Kinetic Studies ◽

Cysteine Residue ◽

Catalytic Site ◽

Steady State Kinetic ◽

State Kinetic

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68922-2 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8845-8849 ◽

Cited By ~ 8

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Reca Protein ◽

Steady State Kinetic ◽

Nucleoside Triphosphates ◽

Hydrolysis Of ◽

State Kinetic

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Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate

Biochemistry ◽

10.1021/bi00660a013 ◽

1976 ◽

Vol 15 (15) ◽

pp. 3244-3253 ◽

Cited By ~ 45

Author(s):

Stephen P. Chock ◽

P. Boon Chock ◽

Evan Eisenberg

Keyword(s):

Steady State ◽

Steady State Kinetic ◽

Myosin Subfragment ◽

Hydrolysis Of ◽

State Kinetic

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A steady-state kinetic study of the ribonuclease A catalyzed hydrolysis of uridine-2′:3′(cyclic)-5′-diphosphate

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(74)90255-0 ◽

1974 ◽

Vol 161 (1) ◽

pp. 227-233 ◽

Cited By ~ 12

Author(s):

Job Ren-Tze Li ◽

Frederick G. Walz

Keyword(s):

Steady State ◽

Kinetic Study ◽

Ribonuclease A ◽

Steady State Kinetic ◽

Hydrolysis Of ◽

State Kinetic

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Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS

Biochemical Journal ◽

10.1042/bj20030204 ◽

2003 ◽

Vol 374 (2) ◽

pp. 369-380 ◽

Cited By ~ 47

Author(s):

Omid HEKMAT ◽

Ken TOKUYASU ◽

Stephen G. WITHERS

Keyword(s):

Steady State ◽

Kinetic Parameters ◽

Intrinsic Rate ◽

Degree Of Polymerization ◽

Colletotrichum Lindemuthianum ◽

Chitin Deacetylase ◽

Steady State Kinetic ◽

Intrinsic Rate Constant ◽

Hydrolysis Of ◽

State Kinetic

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed Km and kcat/Km on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (kcat, 7 s−1) is independent of n and represents the intrinsic rate constant (kint) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed kcat/Km values (A−2, −11.0; A−1, −1.5; A0, −7.7; A+1, −12.5 kJ·mol−1). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [ΔΔG(N-acetyl)=3.3, 0, 4.0 and 0 kJ·mol−1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.

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