Interaction of brain-type creatine kinase with its transition state analog: kinetics of inhibition and conformational changes.
The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The Km for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and the Ki was 0.16 mM; in the presence of the transition state analog, MgADP + NO3- + creatine, the Ki was estimated to be 0.04 mM. Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel filtration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 +/- 0.05 M-1 min-1 to 6.96 +/- 0.11 M-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Grossman
S H
SH
Department of Chemistry, University of South Florida, Tampa 33620.
Garcia-Rubio
L H
LH
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000882:Haplorhini; D007527:Isoenzymes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011487:Protein Conformation
D007527:Isoenzymes; D003402:Creatine Kinase