Effects of pressure on the kinetics of capture by yeast alcohol dehydrogenase

High pressure causes biphasic effects on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase as expressed in the kinetic parameter V/K whic h measures;substrate capture. Moderate pressure increases the rate of captu re of benzyl alcohol by activating:the hydride transfer step. This means th at the transition state for hydride transfer has a smaller volume than the free alcohol plus the capturing form of enzyme, with Delta V double dagger of -39 +/- 1 mL/mol, a value that is relatively large. This is the first ph ysical property of an enzymatic transition state thus characterized, and it offers new possibilities for structure-activity analyses.:Pressures of > 1 .5 kbar decrease the rate of capture of benzyl alcohol by favoring a confor mation of the enzyme which binds nicotinamide adenine:dinucleotide (NAD(+)) ; less tightly. This means that the ground state for tight binding, E*-NAD( +), has;a larger,volume than the collision complex, E-NAD(+), with a Delta V* of 73 +/- 2 mL/mol; The equilibrium: constant of the conformational chan ge K-eq* is 75 +/- 13 at 1 atm. The effects of pressure on the capture of N AD(+) have no activation phase because the conformational change is now bei ng expressed kinetically instead of thermodynamically, together with but in opposition to hydride transfer, causing the effects to cancel. For yeast a lcohol dehydrogenase, this conformational change had not been detected prev iously, but similar conformational changes have been found by spectroscopic means in other dehydrogenases, and, some of-them are also sensitive to pre ssure. The opposite signs for the volume change Of tighter binding and hydr ide transfer run contrary to Pauling's hypothesis that substrates are bound more tightly in the transition state than in the Michaelian reactant state .