Differential Effects of Temperature and Hydrostatic Pressure on the Formation of Quinonoid Intermediates from L-Trp and L-Met by H463F Mutant Escherichia coli Tryptophan Indole-lyase

摘要

Escherichia coli tryptophan indole-lyase (Trpase) is a bacterial pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the reversible beta-elimination of L-Trp to give indole and ammonium pyruvate.H463F mutant E.coli Trpase (H463F Trpase) has very low activity with L-Trp,but it has near wild-type activity with other in vitro substrates,such as S-ethyl-L-cysteine and S-(o-nitrophenyl)-L-cysteine [Phillips,R.S.,Johnson,N.,and Kamath,A.V.(2002) Formation in vitro of Hybrid Dimers of H463F and Y74F Mutant Escherichia coli Tryptophan Indole-lyase Rescues Activity with L-Tryptophan,Biochemistry 41,4012-4019].The interaction of H463F Trpase with L-Trp and L-Met,a competitive inhibitor,has been investigated by rapid-scanning stopped-flow,high-pressure,and pressure jump spectrophotometry.Both L-Trp and L-Met bind to H463F Trpase to form equilibrating mixtures of external aldimine and quinonoid intermediates,absorbing at approx 420 and approx 505 nm,respectively.The apparent rate constant for quinonoid intermediate formation exhibits a hyperbolic dependence on L-Trp and L-Met concentration.The rate constant for quinonoid intermediate formation from L-Trp is approx 10-fold lower for H463F Trpase than for wild-type Trpase,but the rate constant for reaction of L-Met is similar for H463F Trpase and wild-type Trpase.The temperature dependence of the rate constants for quinonoid intermediate formation reveals that both L-Trp and L-Met have similar values of DELTAH~(not=),but L-Met has a more negative value of DELTAS~(not=).Hydrostatic pressure perturbs the spectra of the H463F L-Trp and L-Met complexes,by shifting the position of the equilibria between different quinonoid and external aldimine complexes.Pressure-jump experiments show relaxations at 500 nm after rapid pressure changes of 100-400 bar with both L-Trp and L-Met.The apparent rate constants for relaxation of L-Trp,but not L-Met,show a significant increase with pressure.From these data,the value of DELTAV~(not=) for quinonoid intermediate formation from the external aldimine of L-Trp can be estimated to be -26.5 mL/mol,a larger than expected negative value for a proton transfer.These results suggest that there may be a contribution to the deprotonation reaction either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer associated with the reaction of L-Trp,but not with L-Met.