Redesigning the PheA Domain of Gramicidin Synthetase Leads to a New Understanding of the Enzyme's Mechanism and Selectivity

• Published in: Biochemistry (Easton) 2006-12, Vol.45 (51), p.15495-15504
• Main Authors: Stevens, Brian W, Lilien, Ryan H, Georgiev, Ivelin, Donald, Bruce R, Anderson, Amy C
• Format: Article
• Language: English
• Subjects: Chorismate Mutase - chemical synthesis; Chorismate Mutase - genetics; Chorismate Mutase - metabolism; Escherichia coli Proteins - chemical synthesis; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Kinetics; Multienzyme Complexes - chemical synthesis; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Phenylalanine - chemistry; Phenylalanine - genetics; Phenylalanine - metabolism; Prephenate Dehydratase - chemical synthesis; Prephenate Dehydratase - genetics; Prephenate Dehydratase - metabolism; Protein Binding - genetics; Protein Structure, Tertiary - genetics; Sequence Homology, Amino Acid; Substrate Specificity - genetics; Tryptophan - chemistry; Tyrosine - chemistry; Tyrosine - genetics; Tyrosine - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061788m

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 K D, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by k cat/K M), primarily driven by a 56-fold improvement in K M, 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 K M. 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 K M 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 enzyme:ATP:substrate complex, which is capable of undergoing conformational change.