Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

Journal Article · Tue Jul 26 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00415a017· OSTI ID:6829383

Morrison, J F; Stone, S R

The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/K/sub NADPH/ profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the K/sub i/ profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/K/sub NADPH/, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. The data for deuterium isotope and deuterium solvent isotope effects are consistent with the postulate that, for the reduction of dihydrofolate to tetrahydrofolate, protonation precedes hydride transfer. A scheme is proposed for the indirect transfer of a proton from the enzyme to dihydrofolate.

• Research Organization: Australian National Univ., Canberra City
• OSTI ID: 6829383
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:15; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CATALYSIS
COENZYMES
DEUTERIUM COMPOUNDS
ENZYMES
ESCHERICHIA COLI
HYDROGEN COMPOUNDS
ISOTOPE EFFECTS
KINETICS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PH VALUE
REACTION KINETICS