THE KINETIC CONSEQUENCES OF BINDING OF HEXOKINASE-I TO THE MITOCHONDRIAL OUTER-MEMBRANE

In a number of tissues, a major fraction of the hexokinase isozyme spe cies present is bound at the mitochondrial outer surface. This study a ddresses the kinetic consequences of binding of hexokinase to the oute r membrane of isolated, phosphorylating mitochondria. The primary aim was to separately measure the relative contributions to changes in the kinetic properties of hexokinase which (1) directly result from the b inding as such, and (2) are caused by binding in close proximity to th e site of mitochondrial ATP regeneration. Hexokinase isozyme I was pur ified from rat brain and then bound to intact rat liver mitochondria o r outer membrane vesicles derived from these mitochondria. The apparen t affinity (K-m app) for ATP and the V-max of the bound hexokinase wer e determined by the spectrophotometric measurement of its activity as a function of the ATP concentration in the medium. The data obtained f or the bound enzyme in the two systems were compared to the kinetic ch aracteristics of hexokinase-I present in a non-bound form. Non-bindabl e hexokinase was obtained by mild protease treatment, such that bindab ility was completely abolished while the intrinsic catalytic propertie s remained unaltered. Parallel determinations of the steady-state ATP and ADP levels in mitochondrial suspensions with bound or non-bindable hexokinase present provided additional information on the consequence s of binding. Binding of hexokinase to phosphorylating mitochondria de creased the K-m app for ATP from 0.168 to 0.081 mM while not changing the V-max. IL appeared that both binding per se (K-m app for ATP decre ased from 0.168 to 0.105 mM and from 0.194 to 0.103 mM upon binding to non-phosphorylating mitochondria and outer membrane vesicles, respect ively) and intramitochondrial ATP regeneration (causing a further redu ction in K-m app for ATP from 0.105 to 0.081 mM for the system of phos phorylating mitochondria) jointly contributed to this reduction in K-m app for ATP caused by binding to phosphorylating mitochondria. The ki netic effect exerted by intramitochondrial ATP regeneration persisted in the presence of an excess of extramitochondrial ATP regenerating ac tivity. ATP and ADP measurements in hexokinase-mitochondria: incubatio ns demonstrated that: (i) up to 7 mM ATP in the medium, higher ADP con centrations were maintained for the case of non-bindable enzyme as com pared to the bound enzyme; and (ii) ATP levels were not significantly different and therefore not responsible for the kinetic difference bet ween bound and non-bound hexokinase. The present findings are compatib le with previous suggestions for local channelling of adenine nucleoti des between bound hexokinase and oxidative phosphorylation.