RESUMO
The sequences of genes encoding homologues of 1-aminocyclopropane-1-carboxylate (ACC) synthase, the first enzyme in the two-step biosynthetic pathway of the important plant hormone ethylene, have recently been found in Fugu rubripes and Homo sapiens (Peixoto et al., Gene 246 (2000) 275). ACC synthase (ACS) catalyzes the formation of ACC from S-adenosyl-L-methionine. ACC is oxidized to ethylene in the second and final step of ethylene biosynthesis. Profound physiological questions would be raised if it could be demonstrated that ACC is formed in animals, because there is no known function for ethylene in these organisms. We describe the cloning of the putative human ACS (PHACS) cDNA that encodes a 501 amino acid protein that exhibits 58% sequence identity to the putative Fugu ACS and approximately 30% sequence identity to plant ACSs. Purified recombinant PHACS, expressed in Pichia pastoris, contains bound pyridoxal-5'-phosphate (PLP), but does not catalyze the synthesis of ACC. PHACS does, however, catalyze the deamination of L-vinylglycine, a known side-reaction of apple ACS. Bioinformatic analysis indicates that PHACS is a member of the alpha-family of PLP-dependent enzymes. Molecular modeling data illustrate that the conservation of residues between PHACS and the plant ACSs is dispersed throughout its structure and that two active site residues that are important for ACS activity in plants are not conserved in PHACS.
Assuntos
DNA Complementar/genética , Liases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Frutas/enzimologia , Genes/genética , Humanos , Liases/química , Liases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Plantas/enzimologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
This paper describes the construction, validation and application of an active site model of the serine protease thrombin. Initial use was made of medium resolution X-ray crystallographic structures of thrombin complexed with low molecular weight, non-specific inhibitors to create a computationally useable active site shell of the enzyme. Molecular mechanics methods were then applied to dock known ligands into the active site region in order to derive a model that would accurately predict binding conformations. Validation of the modelling process was achieved by comparison of the predicted enzyme-bound conformations with their known, crystallographic binding conformations. The resultant model was used extensively for predictive purposes prior to obtaining confirmatory crystal data relating to a ligand possessing a novel and unexpected binding component complexed to thrombin. The data served both to confirm the accuracy of the binding site model and to provide information for the further refinement of the model.
Assuntos
Antitrombinas/química , Trombina/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Peso Molecular , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Trombina/antagonistas & inibidoresRESUMO
Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.