Appleman et al., 1990) enables the enzyme to combine Building, University of Leicester, University Road, Leicester LE1 9HN, UK a high affinity for its substrates with a reasonable rate of 1 To whom correspondence should be addressed dissociation of the structurally very similar products, and hence with a reasonable rate of catalytic turnover. Although this kind The effects of six amino acid substitutions in Lactobacillus of mechanism is unusual, it is not unique to DHFR; a similar casei

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... olate reductase, predominantly in the coenmechanism has recently been established for chloramphenicol zyme binding site, on catalysis and on the negative cooperacetyltransferase (Ellis et al., 1995) . ativity between NADPH and tetrahydrofolate binding have A number of structural and mutagenesis studies of L.casei been determined. Replacement of Leu62, His64 or Leu54 and E.coli DHFR have implicated residues in the NADPH by alanine has no effect on k cat , and produces only modest binding site in the mechanism of negative cooperativity changes in negative cooperativity. Alanine substitution of Adams et al., 1989 ; Andrews et al., His77, which interacts indirectly with the coenzyme adenine 1989; Murphy and Bystroff and Kraut, 1991 ; ring, leads to a doubling of the negative cooperativity and Beard et al., 1991; Li et al., 1992; Thomas et al., 1994) . In a consequent doubling of k cat . Replacement of Arg43, which order to explore this further we have constructed various interacts with the coenzyme 2Ј-phosphate, by alanine, or mutations at the NADPH binding site of L.casei DHFR, of Trp21, which interacts with the coenzyme nicotinamide predominantly in or near the adenine sub-site, and compared ring, by histidine leads to a 20-100-fold decrease in negative the kinetic behaviour of the mutant enzymes with the structural cooperativity. In both mutants there is a decrease in k cat ; consequences of the mutation revealed by NMR spectroscopy. isotope effects show that product release is largely ratelimiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1 H NMR has been used to obtain informa-Materials and methods tion on the extent of the structural changes produced by Materials the substitutions. This varies from very local effects in 'Deep Vent' DNA polymerase and the restriction enzymes H64A to very widespread effects in W21H. These changes BamHI and NdeI were purchased from New England Biolabs, are used as the basis for discussion of the mechanisms of Inc. The expression vector pET11a and the E.coli strain BL21 the functional effects of the various substitutions. It is (DE3) pLysS were obtained from Novagen, Inc. 2 H 2 O (99.9% suggested that residues in helix C, β-strand 3 and the β3-2 H) was from Goss Scientific Instruments Ltd. Methotrexate, β4 loop may be involved in the transmission of effects FH 2 , NADP ϩ and 1,N 6 -ethenoadenine dinucleotide phosphate between the coenzyme and substrate binding sites. (εNADP ϩ ) were from Sigma Chemical Co. and were used Keywords: cooperativity/dihydrofolate reductase/NMR/mutants without further purification. NADPH was obtained from Sigma Chemical Co. and was purified prior to use by FPLC using a Pharmacia Mono Q anion exchange column (Orr and Blanch-