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Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate

Published online by Cambridge University Press:  01 November 1999

MENGHSIAO MENG ,
TZE-LIEH CHANE ,
YUH-JU SUN  and
CHWAN-DENG HSIAO
MENGHSIAO MENG
Affiliation:
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung, Taiwan 40227, Republic of China
TZE-LIEH CHANE
Affiliation:
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung, Taiwan 40227, Republic of China
YUH-JU SUN
Affiliation:
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, Republic of China
CHWAN-DENG HSIAO
Affiliation:
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, Republic of China
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Abstract

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in kcat/KM, by a factor of 103, indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 → Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of kcat/KM, supports a role of His311 as a general base in the catalysis.

Keywords

Bacillus stearothermophiluscatalytic mechanismglucose-6-phosphate isomeraseN-bromoacetylethanolamine phosphatephosphoglucose isomerasessite-directed mutagenesis
Type
Research Article
Information
Protein Science , Volume 8 , Issue 11 , November 1999 , pp. 2438 - 2443
Copyright
© 1999 The Protein Society

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