Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: Hydrolyses of thiono and oxygen esters

Bence Asbóth, L. Polgár

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K. H., & Swen, H. M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.

Original languageEnglish
Pages (from-to)117-122
Number of pages6
JournalBiochemistry
Volume22
Issue number1
Publication statusPublished - 1983

Fingerprint

Serine Proteases
Sulfhydryl Compounds
Serine
Hydrolysis
Esters
Peptide Hydrolases
Stabilization
Binding Sites
Oxygen
Sulfur
Catalysis
Chymopapain
Subtilisin
Chemical reactivity
Biochemistry
Papain
Chymotrypsin
Substrates
X-Ray Diffraction
Hydrogen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases : Hydrolyses of thiono and oxygen esters. / Asbóth, Bence; Polgár, L.

In: Biochemistry, Vol. 22, No. 1, 1983, p. 117-122.

Research output: Contribution to journalArticle

@article{3594815fe1c54c9099ee01df36e06abc,
title = "Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: Hydrolyses of thiono and oxygen esters",
abstract = "X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K. H., & Swen, H. M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.",
author = "Bence Asb{\'o}th and L. Polg{\'a}r",
year = "1983",
language = "English",
volume = "22",
pages = "117--122",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases

T2 - Hydrolyses of thiono and oxygen esters

AU - Asbóth, Bence

AU - Polgár, L.

PY - 1983

Y1 - 1983

N2 - X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K. H., & Swen, H. M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.

AB - X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K. H., & Swen, H. M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.

UR - http://www.scopus.com/inward/record.url?scp=0021094368&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021094368&partnerID=8YFLogxK

M3 - Article

C2 - 6338911

AN - SCOPUS:0021094368

VL - 22

SP - 117

EP - 122

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 1

ER -