Trypsin: a case study in the structural determinants of enzyme specificity.
Trypsin and chymotrypsin have similar tertiary structures, although very different substrate specificities. Trypsin hydrolyzes peptides at Lys/Arg residues while chymotrypsin recognizes large hydrophobic residues. Recent work has shown that trypsin is not converted into a protease with chymotrypsin-like activity when the S1 substrate binding site residues are replaced with their chymotrypsin counterparts. Chymotrypsin-like activity is reconstituted in the trypsin framework when two surface loops are substituted with the analogous loops of chymotrypsin in addition to the substitutions at the S1 site. Chymotrypsin-like activity is further improved when Tyr172, another residue located outside the S1 site, is replaced with Trp. These mutant enzymes catalyze the hydrolysis of enzyme-bound substrate at rates comparable to chymotrypsin, but are defective in substrate binding. X-ray crystal structures of the chymotrypsin-like mutants reveal that the loops are disordered and that the Tyr172 to Trp substitution stabilizes the loops. These observations demonstrate that substrate specificity is derived from a network of structural interactions which extends beyond the S1 site.
Hedstrom
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Graduate Department of Biochemistry Brandeis University Waltham, MA 02254, USA.
D000215:Acylation; D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
D014357:Trypsin