KINETIC SCHEME FOR THYMIDYLATE SYNTHASE FROM ESCHERICHIA-COLI - DETERMINATION FROM MEASUREMENTS OF LIGAND-BINDING, PRIMARY AND SECONDARY ISOTOPE EFFECTS, AND PRE-STEADY-STATE CATALYSIS

We have determined kinetic and thermodynamic constants governing bindi ng of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + d UMP --> TS .dUMP + (6R)-5, 10-CH2-H(4)folate --> TS.dUMP.(GR)-5,10-CH( 2)H(4)folate --> TS.dTMP.H(2)folate --> TS .dTMP --> TS as predicted p reviously by others from steady-state measurements. (2)When substrates are saturating, the overall reaction rate is governed by the slow con version of enzyme-bound substrates to bound products as demonstrated b y (i) large primary and secondary isotope effects on k(cat) and (ii) h igh rates of product dissociation compared to k(cat). (3) Stopped-flow studies measuring the binding of 10-propargyl-5,8-dideazafolate, an a nalog of (6R)-5,10-CH(2)H(4)folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physi cal events corresponding to spectral changes which are observed with t he wild-type enzyme during initiation of catalysis. A kinetically iden tifiable reaction step, TS.dUMP.(6R)-5,10-CH(2)H(4)folate --> (TS.dUMP .(6R)-5,10-CH(2)H(4)folate), likely represents reorientation of the C -terminus of the enzyme over the catalytic site. This seals the substr ates into a relatively nonaqueous environment in which catalysis can o ccur. (4) Although TS is a dimer of identical subunits, catalysis is p robably confined to only one subunit at a time, (5) The ''high-resolut ion'' kinetic scheme described herein provides a framework for the int erpretation of the kinetics of catalysis by mutant ecTS chosen to prov ide insights into the relationship between structure and function.