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.

@article{Hussain2003VariationIT,
  title={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.},
  author={S. Hussain and S. Pinitglang and T. S. Bailey and J. D. Reid and M. Noble and M. Resmini and E. Thomas and R. Greaves and C. Verma and K. Brocklehurst},
  journal={The Biochemical journal},
  year={2003},
  volume={372 Pt 3},
  pages={
          735-46
        }
}
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… Expand
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Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.
TLDR
The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants, and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. Expand
The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.
TLDR
The P2/S2 contacts substantially improve enzyme-ligand binding, and greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 and pKa 5, such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and reveal the kinetic influence of a pKa approx. Expand
Evidence for a rate-limiting conformation change in the catalytic steps of the ficin and papain-catalysed hydrolyses of benzyloxycarbonyl-L-lysine p-nitrophenyl ester.
TLDR
Evidence is presented that this “extra”, rate-limiting catalytic step is a conformation change in the enzyme · Z-Lys-ONp complex, prior to the release of p-nitrophenol. Expand
Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.
TLDR
It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites in close agreement with the value determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. Expand
Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain.
TLDR
2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant for its reaction with the catalytic-site thiol group of papain found marked differences in their reactivities towards papain. Expand
A classical enzyme active center motif lacks catalytic competence until modulated electrostatically.
The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of theseExpand
Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.
TLDR
A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state, which accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. Expand
Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.
TLDR
Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the Active Centre aspartic acid carboxy group to influence the reactivity of theActive Centre thiol group at pH values of about 4.4. Expand
DIRECT EVIDENCE FOR AN ACYLATED THIOL AS AN INTERMEDIATE IN PAPAIN- AND FICIN-CATALYSED HYDROLYSES.
TLDR
These experiments provide compelling evidence that the acylation by substrate of both papain and ficin takes place through a thiol residue. Expand
An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.
TLDR
Comparing pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Expand
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