A sequence comparison of nine functionally different GTP-binding protein families has yielded further information on the general characterization ofthe conservation and importance of amino acid sequences in the GTP-binding domain, including (i) a consensus sequence composed of three consensus elements GXXXXGK, DXXG, and NKXD with consensus spacings of either 40-80 or 130-170 amino acid residues between the first and second elements and -40-80 amino acid residues between the second and third sequence elements; (it) the sequence NKXW in place of NKXD in the sequence element responsible for base specificity allows the use of ITP as well as GTP; (fib) dGTP can be used with essentially the same efficiency as GTP; (iv) signal transducing proteins and enzymes have been identified in the nine families; and (v) family conservations allow the identification of the most probable consensus sequence element when more than one is present. Employing these features we have screened the protein sequence data base of the Protein Identification Resource and have identified only known GTP-binding proteins with the exception of protein 2C from foot-and-mouth disease virus as matching the consensus sequence. Based on this finding we predict that foot-and-mouth disease virus protein 2C binds GTP and, by analogy, that protein 2C from several related viruses (polio, rhino, encephalomyocarditis, and cowpea mosaic) will bind a nucleotide as part of its biologic activity.

The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.

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Human chymase and rat chymase-1 are mast cell serine proteases involved in angiotensin II (Ang II) formation and degradation, respectively. Previous studies indicate that both these enzymes have similar P 1 and P 2 preferences, which are the major determinants of specificity. Surprisingly, despite the occurrence of optimal P 2 and P 1 residues at the Phe 82 . The overall effect of this P 1 Ile interaction on catalytic efficiency, however, is influenced by the structure of the acyl group and that of the other leaving group residues. For human chymase, the P 1 Ile interaction is not productive. Thus, specificity for Ang II formation versus Ang II degradation by these chymases is produced through synergistic interactions between acyl or leaving group residues as well as between the acyl and leaving groups. These observations indicate that nonadditive interactions between the extended substrate binding site of human chymase or rat chymase-1 and the substrate are best explained if the entire binding site is taken as an entity rather than as a collection of distinct subsites. Chymases1 are a family of mast cell serine proteinases involved in such diverse functions as inflammation (1), parasite expulsion (2), and peptide hormone processing (3-5). These serine proteinases are synthesized as inactive precursors but are stored in secretory granules as active enzymes (6). Recent phylogenetic evidence indicates that mammalian chymases occur as two distinct isoenzyme groups, ␣ and ␤ (7). ␣-Chymases include human chymase, dog chymase, mouse chymase-5, rat chymase-3, and gerbil chymase-2 (7, 8). ␤-Chymases include rat chymase-1 and -2, mouse chymase-1, -2, -4, and -L, and gerbil chymase-1 (7,8 . Early comparative studies on chymase specificities by Powers et al. (11) using peptide 4-nitroanilide substrates indicated that the S 1 to S 4 subsites 3 of human chymase and rat chymase-1 are similar. In both human chymase and rat chymase-1, the key features for optimal acyl group interactions are a P 1 hydrophobic aromatic residue, a P 2 hydrophobic residue or Pro, and a P 3 hydrophobic residue. S 4 subsite interactions are less restrictive. Thus, these studies could not explain why human chymase is an Ang II-forming enzyme and rat chymase-1 is an angiotensinase (3, 5) and suggested to us that enzyme-substrate interactions other than those occurring at the S 1 to S 4 subsites of these enzymes could be important for determining specificity. In a previous paper we explored the S 1 subsite as well as the SЈ 1 to SЈ 2 subsites of human chymase using decapeptide Ang I analogs. We showed that a P 1 hydrophobic aromatic residue was necessary but that several non-* This work was supported by National Institutes of Health Grant HL44201. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed: Dept. of Molecular Cardiology, FF30,...

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.