Redesigning the PheA domain of gramicidin synthetase leads to a new understanding of the enzyme's mechanism and selectivity.
Biochemistry
; 45(51): 15495-504, 2006 Dec 26.
Article
em En
| MEDLINE
| ID: mdl-17176071
ABSTRACT
The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectively binds phenylalanine along with ATP and Mg2+ and catalyzes the formation of an aminoacyl adenylate. In this study, we have used a novel protein redesign algorithm, K*, to predict mutations in PheA that should exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutations to PheA did not significantly improve KD, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by kcat/KM), primarily driven by a 56-fold improvement in KM, although the improvement did not make tyrosine the preferred substrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different amino acid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand the improvement in KM. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-type enzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared to bind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA, phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalanine complex may undergo a conformational change toward a catalytically relevant intermediate on the pathway to adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step binding process. The improvement in KM for the mutant enzyme toward tyrosine, as predicted by K*, may indicate that redesigning the side-chain binding pocket allows the substrate backbone to adopt productive conformations for catalysis but that further improvements may be afforded by modeling an enzymeATPsubstrate complex, which is capable of undergoing conformational change.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Prefenato Desidratase
/
Corismato Mutase
/
Estrutura Terciária de Proteína
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Proteínas de Escherichia coli
/
Complexos Multienzimáticos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Estados Unidos