Active-site titration of enzymes at high concentration. Application to myosin ATPase

The number of active sites of soluble and filamentous myosin and of its subfragments, heavy meromyosin and subfragment-1, has been determined. The titration involves steady-state kinetic measurements at a high enzyme concentration and varying substrate concentrations (or vice versa), in the presence of a substrate-regenerating system. Some practical and theoretical conditions for its execution are given, and, in particular, the effect of a possible heterogeneity of the active sites on the titration curves is analysed. Under the experimental conditions of the study, the number of active sites is close to that of myosin heads, and the heads seem to be functionally identical; the catalytic constants kcat and Km characterizing each active site are similar within some limits (1-2 for the ratio of kcat values; 1-5 for that of Km values).