Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site.
We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) with the active site-directed reagent bromo[U-14C]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 14C-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 [O'Brien, W. E., McInnes, R., Kalumuck, K. & Adcock, M. (1986) Proc. Natl. Acad. Sci. USA 83, 7211-7215; Beacham, I. R., Schweitzer, B. W., Warrick, H. M. & Carbon, J. (1984) Gene 29, 271-279]. The 14C-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.
D000595:Amino Acid Sequence; D000818:Animals; D001123:Argininosuccinate Lyase / Q000378:metabolism*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002417:Cattle; D005650:Fumarates / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008190:Lyases / Q000378:metabolism*; D008239:Lysine; D010446:Peptide Fragments / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D014357:Trypsin
D002250:Carbon Radioisotopes; D005650:Fumarates; D010446:Peptide Fragments; C033312:bromomesaconic acid; D014357:Trypsin; D008190:Lyases; D001123:Argininosuccinate Lyase; D008239:Lysine