Inactivation of brain hexokinase by an adenosine 5'-triphosphate analog

Subbarao, B. ; Kenkare, Umakant W. (1977) Inactivation of brain hexokinase by an adenosine 5'-triphosphate analog Archives of Biochemistry and Biophysics, 181 (1). pp. 19-25. ISSN 0003-9861

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/000398...

Related URL: http://dx.doi.org/10.1016/0003-9861(77)90478-7

Abstract

An analog of ATP, 6-mercapto-9-β-D -ribofuranosylpurine 5'-triphosphate (SH-TP), functions as a phosphoryl donor for the reaction catalyzed by hexokinase. Incubation of the enzyme with millimolar concentrations of this reagent led to its rapid inactivation. However, the corresponding monophosphate derivative of this reagent was much less effective. A plot of the initial rate of inactivation versus the concentration of SH-TP exhibited saturation, suggesting the formation of a reversible complex between the enzyme and SH-TP prior to the inactivation reaction. The dissociation constant of this enzyme-inhibitor complex was the same as the Km of this reagent with respect to hexokinase in the phosphoryl transfer reaction. These data and protection by the substrates glucose, ATP, and ITP against this inactivation indicate that SH-TP is an active site-directed reagent for hexokinase. The reactivation of the inactivated enzyme by reducing thiol reagents and the disappearance of free thiols after reaction with SH-TP show that sulfhydryl groups of the enzyme are those modified. However, no enzyme bound SH-TP could be demonstrated. Progress curves of inactivation of the enzyme by SH-TP, plotted in a first-order fashion, showed biphasicity. These results are explained by a proposal that two thiols, one essential and the other nonessential, lie in close proximity at the active site of hexokinase.

Item Type:Article
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ID Code:16223
Deposited On:15 Nov 2010 14:01
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