Abstract：
:The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family features several l-aminoacylases useful in biocatalysis. Mammalian Acy1, in particular, has been applied in racemic resolution and reverse hydrolysis. Despite recent advances in our understanding of the active site architecture and functioning, determinants of Acy1 substrate specificity have remained uncharted. Comparison to bacterial homologues points to a sterically more restricted acyl-binding pocket for Acy1. Here we sought to map characteristics of the acyl-binding pocket of human and porcine Acy1. Toward this end, we determined Michaelis constants for an analogue series of aliphatic N-acyl- l-methionine substrates and translated the values into three-dimensional quantitative structure-activity relationship models employing the minimal topological difference-partial least square method. The QSAR models for the two enzymes suggest overall similar binding pockets in the acetyl-binding portion and indicate a general preference for straight-chain acyl moieties. Embedding of the QSAR map for human Acy1 in the structure of its metal-binding domain associates the side chain of Ile177 with limited acyl chain elongation which was not observed for the porcine enzyme. The topological model further supports roles of Thr347 and Leu372, which are both conserved in the porcine enzyme, in restricting acyl chain branching at the alpha- and beta-positions, respectively. Mutational analyses confirmed our predictions for Thr347 and Leu372. Moreover, the T347S variant of human Acy1 exhibited markedly increased catalytic efficiency against N-benzoylamino acids, demonstrating the potential for engineering of substrate specificity in Acy1. We discuss the more general application of the employed procedure for protein design.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Lindner HA,Alary A,Wilke M,Sulea Tdoi
10.1021/bi702156hsubject
Has Abstractpub_date
2008-04-08 00:00:00pages
4266-75issue
14eissn
0006-2960issn
1520-4995journal_volume
47pub_type
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