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

Abstract

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 substrateregenerating 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 k,,, and K, characterizing each active site are similar within some limits (1 -2 for the ratio of k,,, values; 1 -5 for that of K , values).The determination of the absolute concentration of enzymes requires the titration of their active sites [l]. Ths is usually possible by combined steady-and presteady-state kinetic measurements, if an enzyme-bound intermediate accumulates during the reaction of the enzyme with its substrate and if a reaction product is concomitantly released. In the case where no intermediate accumulates, this active-site titration is not applicable.An alternative method involving steady-state kinetic measurements may be used if the apparent affinity of the enzyme for its substrate is high. In this case, the rate of the reaction at high enzyme concentration is maximum when the substrate concentration becomes equal to that of the concentration of enzyme active sites. Although this possibility has been known for a long time [2], it seems to have been hardly used, to our knowledge, except for a recent application to the active-site titration of soluble myosin and of its subfragments [3-61. This last study was simplified particularly by the regeneration of the substrate during the course of experiments due to an efficient substrate-regenerating system. In the present paper, we develop some theoretical and practical conditions for this titration as well as some pitfalls which may arise when it is carried out.Muscle myosins are double molecules made up of one pair of heavy chains and two pairs of light chains, the so-called alkali and regulatory subunits. In the case of fast-twitch muscle myosins, there are two different alkali subunits, the A1 and A2 chains, which have however no distinct effect on of two different heavy chains in an approximate ratio of 60:40. At the present time, the number and the identity of active sites of the two-headed myosin are still under debate [3,9 -121 and the question of the functional homogeneity or heterogeneity of these sites is not yet resolved. These points will be discussed from the results of active-site titrations of myosin and of its subfragments. THEORYSeveral authors have discussed steady-state enzyme kinetics with high concentrations of substrate and enzyme [2, 13 -161. For the following general enzyme reaction mechanism : E + S e E S .+ E + P , the initial rate ...

Synergistic Binding of the Phosphorylated S233- and S259-Binding Sites of C-RAF to One 14-3-3ζ Dimer

“…To validate this finding, we performed an active site titration. 28 In this experiment, 3 μM di-phosphorylated FAM-CRAFpS233,pS259 peptide (10-fold excess over K d ) was titrated with 14-3-3ζ from two different starting concentrations (Fig. 1d).…”

Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits

“…Mg2+-dependent ATPase activities and active site titrations were spectrophotometrically measured by means of the ADP-linked assay system under the conditions previously described [9]. Fluorescence stopped-flow experiments were performed according to Tesi et al [lo], while kinetic results were analysed according to Biosca et al [ll], using the Bagshaw-Trentham scheme [12] (Scheme 1):…”

Production and Purification of Large Quantities of the Functional N-Terminal Ectodomain of Human VPAC1 Receptor

“…Briefly, intrinsic fluorescence of tryptophan residues (W 67 , W 73 , and W 110 ) from the purified N-ted was measured in 2 ml of hydroxyethylpiperazine ethane sulfonic buffer pH 7.5 containing 1 μM purified N-ted, in absence or presence of increasing concentration (up to 9 μM) of natural agonist (VIP) or antagonist (PG97-269). Dissociation constants were determined from titration curves using analytical procedure developed by Bechet et al (1986).…”