Transition-state characterization

A new approach combining inhibitor analogues and variation in enzyme structure

Margaret A. Phillips, Alan P. Kaplan, William J. Rutter, Paul A. Bartlett

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.

Original languageEnglish (US)
Pages (from-to)959-963
Number of pages5
JournalBiochemistry®
Volume31
Issue number4
StatePublished - 1992

Fingerprint

Organophosphonates
Guanidine
Enzymes
Carboxypeptidases A
Dipeptides
Substrates
Cations
Rats
Hydrolysis
Rate constants
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition-state characterization : A new approach combining inhibitor analogues and variation in enzyme structure. / Phillips, Margaret A.; Kaplan, Alan P.; Rutter, William J.; Bartlett, Paul A.

In: Biochemistry®, Vol. 31, No. 4, 1992, p. 959-963.

Research output: Contribution to journalArticle

Phillips, Margaret A. ; Kaplan, Alan P. ; Rutter, William J. ; Bartlett, Paul A. / Transition-state characterization : A new approach combining inhibitor analogues and variation in enzyme structure. In: Biochemistry®. 1992 ; Vol. 31, No. 4. pp. 959-963.
@article{804e60a37487488f9b6ba388a1b2aede,
title = "Transition-state characterization: A new approach combining inhibitor analogues and variation in enzyme structure",
abstract = "A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.",
author = "Phillips, {Margaret A.} and Kaplan, {Alan P.} and Rutter, {William J.} and Bartlett, {Paul A.}",
year = "1992",
language = "English (US)",
volume = "31",
pages = "959--963",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "4",

}

TY - JOUR

T1 - Transition-state characterization

T2 - A new approach combining inhibitor analogues and variation in enzyme structure

AU - Phillips, Margaret A.

AU - Kaplan, Alan P.

AU - Rutter, William J.

AU - Bartlett, Paul A.

PY - 1992

Y1 - 1992

N2 - A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.

AB - A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.

UR - http://www.scopus.com/inward/record.url?scp=0026581791&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026581791&partnerID=8YFLogxK

M3 - Article

VL - 31

SP - 959

EP - 963

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 4

ER -