Modification of a thiol at the active site of the Ascaris suum NAD-malic enzyme results in changes in the rate-determining steps for oxidative decarboxylation of L-malate

Journal Article · Tue Jun 11 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00237a019· OSTI ID:5081054

Gavva, S R; Harris, B G; Cook, P F ^[1]; Weiss, P M ^[2]

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1. Texas Coll. of Osteopathic Medicine, Fort Worth (United States)
2. Univ. of Wisconsin, Madison (United States)

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K{sub NAD} and K{sub i metal} and decreases in V{sub max} as the metal size increases from Mg{sup 2+} to Mn{sup 2+} to Cd{sup 2+}, indicative of crowding in the site. The K{sub malate} value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K{sub malate} increase with all three metal ions compared to the native enzyme concomitant with a decrease in the {sup 13}C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The {sup 13}C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary {sup 13}C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd{sup 2+}. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd{sup 2+} as the divalent metal activator. Upon inactivation, only Cd{sup 2+} ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd{sup 2+}.

• OSTI ID: 5081054
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:23; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ASCARIDAE
ASCARIS
ASCHELMINTHES
BIOCHEMICAL REACTION KINETICS
CADMIUM COMPOUNDS
CARBON 13
CARBON ISOTOPES
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLIC ACIDS
CATIONS
CHARGED PARTICLES
CHEMICAL REACTIONS
DECARBOXYLASES
DECARBOXYLATION
ENZYMES
EVEN-ODD NUCLEI
HELMINTHS
HYDROXY ACIDS
INACTIVATION
IONS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
MALIC ACID
NEMATODES
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
REACTION KINETICS
STABLE ISOTOPES
THIOLS