A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

Abstract

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

“…31 Another ligand, the tetrahydrofolate analogue 5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), binds to DHFR with strong negative co-operativity in the presence of NADPH: 32 similar negative co-operativity binding effects have important implications in the control of product release in the enzyme reaction mechanism. 33 The detailed origins of positive and negative co-operative ligand binding are still poorly understood. One possible explanation calls for the first ligand to induce conformational changes in the protein that produce an altered binding environment for the second ligand.…”

Domain motions in dihydrofolate reductase: a molecular dynamics study

“…While the sequence homology between different vertebrate dhfrs is high (73 to 90%), that between the enzymes from Lactobacillus casei and Escherichia coli is only about 26%. Nonetheless, the overall backbone folds for the two bacterial enzymes, as judged from the crystal structures, are almost identical (Kraut & Matthews, 1987), as indeed are their kinetic mechanisms (Andrews et al, 1989). Notwithstanding these extensive studies of dhfr, a number of aspects of its function are still not completely understood, notably the details of the catalytic mechanism (Basran et al, 1995 and references therein) and the mechanism(s) underlying the effects of substrate analogues on the binding of coenzymes and vice versa.…”

“…see Neet, 1995 and references therein). There is also a marked negative cooperativity between NADPH and the product tetrahydrofolate (FH 4 ) or its stable 5-formyl analogue (Birdsall et al, 1981), the increased rate of dissociation of FH 4 from the enzyme-NADPH-FH 4 complex as compared to the enzyme-FH 4 complex being an important feature of the kinetic mechanisms of both the L. casei and E. coli enzymes (Fierke et al, 1987;Andrews et al, 1989).…”

NMR Studies of Ligand Binding to Dihydrofolate Reductase

“…Benkovic et al [28,29,56,57] and Blakley et al [58] have also carried out extensive kinetic studies on several DHFRs. For example, Andrews, Benkovic, and co-workers [56] have shown that for the L. casei enzyme, the dissociation of the product is the rate-limiting step for the steady-state turnover at low pH values.…”

“…For example, Andrews, Benkovic, and co-workers [56] have shown that for the L. casei enzyme, the dissociation of the product is the rate-limiting step for the steady-state turnover at low pH values. The product, tetrahydrofolate, only dissociates from the enzyme after NADP is replaced by NADPH.…”