Active-site variants of Streptomyces griseus protease B with peptide-ligation activity.
BACKGROUND
Peptide-ligating technologies facilitate a range of manipulations for the study of protein structure and function that are not possible using conventional genetic or mutagenic methods. To different extents, the currently available enzymatic and nonenzymatic methodologies are synthetically demanding, sequence-dependent and/or sensitive to denaturants. No single coupling method is universally applicable. Accordingly, new strategies for peptide ligation are sought.


RESULTS
Site-specific variants (Ser195-->Gly, S195G, and Ser195-->Ala, S195A) of Streptomyces griseus protease B (SGPB) were generated that efficiently catalyze peptide ligation (i.e., aminolysis of ester-, thioester- and para-nitroanilide-activated peptides). The variants also showed reduced hydrolytic activity relative to the wild-type enzyme. The ratio of aminolysis to hydrolysis was greater for the S195A variant, which was also capable of catalyzing ligation in concentrations of urea as high as 2 M.


CONCLUSIONS
Mutagenic substitution of the active-site serine residue of SGPB by either glycine or alanine has created a unique class of peptide-ligating catalysts that are useful for coupling relatively stable ester- and para-nitroanilide-activated substrates. Ligation proceeds through an acyl-enzyme intermediate involving His57. Serine to alanine mutations may provide a general strategy for converting proteases with chymotrypsin-like protein folds into peptide-coupling enzymes.
Elliott
R J
RJ
Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, V5A 1S6, Canada.
Bennet
A J
AJ
Braun
C A
CA
MacLeod
A M
AM
Borgford
T J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D017931:DNA Primers; D009842:Oligopeptides; D012697:Serine Endopeptidases; C024984:streptogrisin B