Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine

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Abstract
Concomitant with the formation of the transition state analog complex [rabbit skeletal muscle] creatine kinase .cntdot. ADPMg .cntdot. nitrate .cntdot. creatine there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure. The kinetics of formation and final amplitude of the spectrum were studied at -15.degree. C using a stopped-flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme-ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine. The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions. This comparison revealed a striking analogy between the 2 processes. The conformation of creatine kinase in the analog complex may be very similar to that in the transition state complex on the catalytic pathway.