Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

Journal Article · Tue Dec 29 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00400a015· OSTI ID:7000064

Howell, E E; Warren, M S; Booth, C L; Villafranca, J E; Kraut, J

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.

• Research Organization: Agouron Institute, La Jolla, CA (USA)
• OSTI ID: 7000064
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:26; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANTIMETABOLITES
BACTERIA
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
DEUTERIUM
DRUGS
ELEMENTARY PARTICLES
ENZYMES
ESCHERICHIA COLI
FERMIONS
GENES
HADRONS
HYDROGEN ISOTOPES
HYDROXY ACIDS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
METHOTREXATE
MICROORGANISMS
MUTANTS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PROTONS
REACTION KINETICS
SERINE
STABLE ISOTOPES
STRUCTURE-ACTIVITY RELATIONSHIPS