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 language | English (US) |
|---|---|
| Pages (from-to) | 485-492 |
| Number of pages | 8 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 316 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1995 |
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ASJC Scopus subject areas
- Molecular Biology
- Biophysics
- Biochemistry
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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 journal › Article
}
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.
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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 -