RESUMEN
The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.
Asunto(s)
Lipasa/química , Lipasa/metabolismo , Mutación/genética , Desnaturalización Proteica , Pseudomonas fragi/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Caprilatos/metabolismo , Estabilidad de Enzimas , Hidrólisis , Cinética , Lipasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Pseudomonas fragi/genética , Alineación de Secuencia , Especificidad por Sustrato , Temperatura , Triglicéridos/metabolismoRESUMEN
The multi-domain protein hSos1 plays a major role in cell growth and differentiation through its Ras-specific guanine nucleotide exchange domain whose complex regulation involves intra-molecular, inter-domain rearrangements. We present a stochastic mathematical model describing intra-molecular regulation of hSos1 activity. The population macroscopic effect is reproduced through a Monte-Carlo approach. Key model parameters have been experimentally determined by BIAcore analysis. Complementation experiments of a Saccharomyces cerevisiae cdc25(ts) strain with Sos deletion mutants provided a comprehensive data set for estimation of unknown parameters and model validation. The model is robust against parameter alteration and describes both the behavior of Sos deletion mutants and modulation of activity of the full length molecule under physiological conditions. By incorporating the calculated effect of amino acid changes at an inter-domain interface, the behavior of a mutant correlating with a developmental syndrome could be simulated, further validating the model. The activation state of Ras-specific guanine nucleotide exchange domain of hSos1 arises as an "emergent property" of its multi-domain structure that allows multi-level integration of a complex network of intra- and inter-molecular signals.