THE PROTHROMBINASE REACTION - MECHANISM SWITCHING BETWEEN MICHAELIS-MENTEN AND NON-MICHAELIS-MENTEN BEHAVIORS

Kinetic properties of prothrombinase were investigated as a function o f composition and structure of the membrane component. The kinetic pro perties were quite diverse, giving linear or nonlinear Eadie-Hofstee p lots and substrate concentrations at half-maximum velocity ([S](0.5)) that varied from 5 to more than 200 nM. This reaction might be describ ed as a ''catalytic system'' in order to distinguish it from standard models that have been developed to describe the kinetics of soluble en zymes, The latter do not anticipate a key feature of prothrombinase an d probably other membrane-bound enzymes, which is the presence of reac tion steps that do not contain an enzyme (E) term. At least four kinet ic mechanisms can arise from a logical series of steps that may occur during the prothrombinase reaction. All of these mechanisms appeared t o contribute to reaction properties under some conditions. In some cas es, one mechanism dominated at low substrate concentration and another at high substrate concentration. This change in the course of a titra tion was referred to as ''mechanism switching'' Only membranes of low phosphatidylserine (PS) content displayed Michaelis-Menten behavior. T ransfer of substrate from the membrane surface to the enzyme was not i mportant so that the enzyme was involved in capture of substrate direc tly from solution. As PS content increased, transfer of substrate from the membrane surface to the enzyme occurred. In these cases, multiple mechanisms contributed to the reaction so that KM and apparent K-M, p roperties that describe an enzyme active site, were not appropriate, e ven when Eadie-Hofstee plots were linear. At high PS content, the enzy me captured every substrate molecule that became bound to the same ves icle. Reaction velocity was governed entirely by protein-membrane bind ing rather than by enzyme properties. Eadie-Hofstee plots were often n onlinear and/or V-max was less than k(cat)[E(t)]. A small impact from collision-limited kinetics was also detected. Small unilamellar vesicl es (SUV, 30 nm diameter) gave higher [S](0.5) values than large unilam ellar vesicles (LUV, 100 nm diameter) of the same phospholipid composi tion. There appeared to be two bases for this behavior. First, LUV may provide a better relationship between the phospholipid surface and th e enzyme, giving a better substrate binding site. Second, for membrane s containing high PS, the number of substrate binding sites per vesicl e contributed to the enhanced function of LUV. These studies showed th at mechanism-switching was important to prothrombinase reaction in vit ro and suggest that various mechanisms, generated by the nature of the membrane, may be an important regulator for prothrombinase behavior i n vivo.