Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

Journal Article · Tue Jan 14 00:00:00 EST 1986 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00349a032· OSTI ID:5528966

Kiick, D M; Harris, B G; Cook, P F

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. Results indicate that substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.

• Research Organization: North Texas State Univ., Denton
• OSTI ID: 5528966
• Journal Information: Biochemistry; (United States), Vol. 1
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
DEUTERIUM
ISOTOPE EFFECTS
NAD
BIOCHEMICAL REACTION KINETICS
ASCARIS
ENZYME ACTIVITY
MITOCHONDRIA
PH VALUE
SUBSTRATES
ASCARIDAE
ASCHELMINTHES
CELL CONSTITUENTS
COENZYMES
HELMINTHS
HYDROGEN ISOTOPES
ISOTOPES
KINETICS
LIGHT NUCLEI
NEMATODES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PARASITES
REACTION KINETICS
STABLE ISOTOPES
560111* - Radiation Effects on Biochemicals- In Vitro- (-1987)