Proteolytic dissection of rat brain hexokinase: Determination of the cleavage pattern during limited digestion with trypsin

Abstract

Limited treatment of rat brain hexokinase (ATP: d-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the M r 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of M r 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as ▪ where T 1 and T 2 indicate tryptic cleavage sites; cleavage at only T 1 or T 2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (~20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the K m for Glc or the K i for Glc-6-P, though a slight decrease in K m for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.