Wheatgerm Hexokinase: Physical and Active‐Site Properties

Abstract

The L II form of wheatgerm hexokinase has been purified and is homogeneous by criteria of electrophoresis and ultracentrifugation. The purified enzyme has a specific activity of 3.5 units/mg with respect to glucose and is a single polypeptide chain of 51000 molecular weight. On the basis of K m values, fructose appears to be the preferred hexose substrate in contrast to the hexokinases of mammals and yeast. Data from “dead‐end” inhibition studies suggest that a rapid‐equilibrium random mechanism obtains but end‐product inhibition indicates that the mechanism may show some additional complexities. The pH dependence of the alkylation and inactivation of the enzyme by iodoacetamide indicates the participation of a group of p K a 6.58 in these processes. This group is not itself alkylated, but its state of protonation determines the rate of the alkylation reaction. Substrates and products affect the rate of alkylation, and the dissociation constants of the enzyme · substrate and enzyme product · complexes are similar to the K m and K i values for these ligands. These data also are consistent with a random mechanism. Studies on the pH dependence of photo‐oxidation of the enzyme show a bisphasic loss of enzyme activity with respect to time. Two ionizing groups with p K a values of 6.78 and 7.44 are involved in the first and second phases, respectively. Investigation of the effects of ligands on the rate of photooxidation shows that, for the first phase, the values for the dissociation constants are equal to the K m and K i values for the substrates and products. However, for the second phase the values are greatly elevated. This may imply that a conformation change occurs in the enzyme following the initial phase of photo‐oxidation. A possible role for hexokinases in germinating cereal seeds is discussed.