N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase

Y. C. Huang, R. J. Haselbeck, L. Mcalisterhenn, R. F. Colman

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Original languageEnglish (US)
Pages (from-to)485-492
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume316
Issue number1
DOIs
StatePublished - Jan 1995

Fingerprint

Isocitrate Dehydrogenase
Ethylmaleimide
NADP
Yeast
Yeasts
Swine
Binding Sites
Adenine
Cysteine
Metals
Escherichia coli
Crystal structure
isocitrate dehydrogenase (NADP+)
maleimide
isocitric acid

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase. / Huang, Y. C.; Haselbeck, R. J.; Mcalisterhenn, L.; Colman, R. F.

In: Archives of Biochemistry and Biophysics, Vol. 316, No. 1, 01.1995, p. 485-492.

Research output: Contribution to journalArticle

Huang, Y. C. ; Haselbeck, R. J. ; Mcalisterhenn, L. ; Colman, R. F. / N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase. In: Archives of Biochemistry and Biophysics. 1995 ; Vol. 316, No. 1. pp. 485-492.
@article{376dc46cb94541a08b99939d4cacb0a3,
title = "N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase",
abstract = "Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50{\%} of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.",
author = "Huang, {Y. C.} and Haselbeck, {R. J.} and L. Mcalisterhenn and Colman, {R. F.}",
year = "1995",
month = "1",
doi = "10.1006/abbi.1995.1064",
language = "English (US)",
volume = "316",
pages = "485--492",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase

AU - Huang, Y. C.

AU - Haselbeck, R. J.

AU - Mcalisterhenn, L.

AU - Colman, R. F.

PY - 1995/1

Y1 - 1995/1

N2 - Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

AB - Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

UR - http://www.scopus.com/inward/record.url?scp=0028984997&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028984997&partnerID=8YFLogxK

U2 - 10.1006/abbi.1995.1064

DO - 10.1006/abbi.1995.1064

M3 - Article

C2 - 7840654

AN - SCOPUS:0028984997

VL - 316

SP - 485

EP - 492

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -