RESUMO
Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.
Assuntos
Inibidores de Cisteína Proteinase/química , Isoenzimas/química , Leupeptinas/química , Estrutura Quaternária de Proteína , Serina Endopeptidases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leupeptinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , TriptasesRESUMO
Human mast cell tryptases represent a subfamily of trypsin-like serine proteinases implicated in asthma. Unlike beta-tryptases, alpha-tryptases apparently are proteolytically inactive. We have solved the 2.2A crystal structure of mature human alpha1-tryptase. It reveals a frame-like tetrameric architecture that, surprisingly, does not require heparin-binding for stability. In marked contrast to beta2-tryptase, the Ser214-Gly219 segment, which normally provides the template for substrate binding, is kinked in alpha-tryptase, thereby blocking its non-primed subsites. This so far unobserved subsite distortion is incompatible with productive substrate binding and processing. alpha-Tryptase apparently is trapped in this off-conformation by repulsions and attractions of the Asp216 side-chain. However, proteolytic activity could be generated by an induced-fit mechanism.
