Combination of 31P-NMR magnetization transfer and radioisotope exchange methods for assessment of an enzyme reaction mechanism: rate-determining steps of the creatine kinase reaction.

The theoretical analysis of a reversible enzyme reaction performed in this work shows that the 31P-NMR magnetization (saturation) transfer technique combined with a radioisotope exchange method may potentially provide information on the position of rate-determining step(s). It depends on chemical shifts of NMR signals of nuclei of interest in free and enzyme-bound forms of substrate(s) and product(s) of the reaction. The creatine kinase reaction (MgATP + creatine----MgADP + P-creatine) has been used as a model. Chemical shifts of 31P in binary, ternary and transitional state substrate-enzyme complexes have been estimated by the variable frequency saturation transfer (VFST) method. This method is based on selective irradiation of numerous points in the spectrum and observation of changes in the intensity of visible line(s) which occur due to chemical exchange between it and lines which are not visible in the routine spectrum. Also, dissociation rate constants of MgADP-containing complexes were determined. Magnetization exchange rates, P-creatine----[gamma-P]MgATP and [beta-P]MgADP----[beta-P]MgATP, were compared with radioisotope exchange rates, [gamma-32P-MgATP----P-creatine and [3H]MgADP----MgATP at different [P-creatine]/[creatine] ratios and at different temperatures. All these exchange rates were close to each other at 30-37 degrees C and [PCr]/[Cr] ratios lower than 2. It is concluded that phosphoryl group transfer is the rate-determining step of the overall creatine kinase reaction under these conditions. However, at lower temperatures (below 25 degrees C) or at high [PCr]/[Cr] ratios ([ADP] less than 20 microM) the rate-determining step seems to be shifted toward dissociation of nucleotide substrates from enzyme-substrate complexes, since exchange rates became significantly different. This approach is useful for analysis of mechanism of enzymatic reactions and also can be applied to non-enzymatic reactions and evaluation of small rapidly exchangeable metabolite pools.