Yeast hexokinase A. Succinylation and properties of the active subunit.
Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC2.7.1.1) dissociates into its subunits upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the some order as those found for the native enzyme. Of the 37 amino groups present per enzyme subunit, 2-3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50%loss of the enzyme activity may be related to the dissociation of the dimer into monomers. Both native enzyme and the succinylated subunits have the same H-dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form. The succinylated subunit presents 'negative co-operativity' with respect to ATP at slightly acid pH; however, the burst-type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.
D000215:Acylation; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002951:Citrates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005994:Glycerophosphates / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008293:Malates / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013386:Succinates; D013438:Sulfhydryl Compounds / Q000032:analysis
D002951:Citrates; D005994:Glycerophosphates; D046911:Macromolecular Substances; D008293:Malates; D010710:Phosphates; D013386:Succinates; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D006593:Hexokinase; D008274:Magnesium