IDENTIFICATION OF A PHOSPHATE REGULATORY SITE AND A LOW-AFFINITY BINDING-SITE FOR GLUCOSE-6-PHOSPHATE IN THE N-TERMINAL HALF OF HUMAN BRAINHEXOKINASE

Crystal structures of human hexokinase I reveal identical binding site s for phosphate and the g-phosphoryl group of glucose B-phosphate in p roximity to Gly(87), Ser(88), Thr(232), and Ser(415), a binding site f or the pyranose moiety of glucose B-phosphate in proximity to Asp(84), Asp(413), and Ser(449), and a single salt link involving Arg(801) bet ween the N- and C-terminal halves. Purified wildtype and mutant enzyme s (Asp(84) --> Ala, Gly(87) --> Tyr, Ser(88) --> Ala, Th-232 --> Ala, Asp(413) --> Ala, Ser(415) --> Ala, Ser(449) --> Ala, and Arg(801) --> Ala) were studied by kinetics and circular dichroism spectroscopy. Al l eight mutant hexokinases have k(cat) and K-m values for substrates s imilar to those of wild-type hexokinase I. Inhibition of wild-type enz yme by 1,5-anhydroglucitol B-phosphate is consistent with a high affin ity binding site (K-i = 50 mu M) and a second, low affinity binding si te (K-ii = 0.7 mM), The mutations of Asp(84), Gly(87), and Thr232 list ed above eliminate inhibition because of the low affinity site, but no ne of the eight mutations influence K-i of the high affinity site. Rel ief of 1,5-anhydroglucitol B-phosphate inhibition by phosphate for Asp (84) --> Ala, Ser(88) --> Ala, Ser(415) --> Ala, Ser(449) --> Ala and Arg(801) --> Ala mutant enzymes is substantially less than that of wil d-type hexokinase and completely absent in the Gly(87) --> Tyr and Thr (232) --> Ala mutants. The results support several conclusions. (i) Th e phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose B-phosphate binding site at th e N-terminal domain is a low affinity site and not the high affinity s ite associated with potent product inhibition. (iii) Arg(801) particip ates in the regulatory mechanism of hexokinase I.