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Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site

Journal Article · · Proc. Natl. Acad. Sci. U.S.A.; (United States)
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We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase with the active site-directed reagent bromo(U-/sup 14/C)mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two /sup 14/C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources The /sup 14/C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis.

Research Organization:
Public Health Research Institute, Inc., New York, NY
OSTI ID:
6409464
Journal Information:
Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 10; ISSN PNASA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKYLATION
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CATTLE
CHEMICAL REACTIONS
DICARBOXYLIC ACIDS
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
FUMARIC ACID
FUNGI
GLANDS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
LYASES
LYSINE
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
PLANTS
REACTION KINETICS
RUMINANTS
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
SERINE PROTEINASES
SUCCINIC ACID
TRACER TECHNIQUES
TRYPSIN
VERTEBRATES
YEASTS