Modulation of Enzyme Specificity by Site-Directed Mutagenesis

Lizbeth Hedstrom, Laszlo Graf, Caro Beth Stewart, William J. Rutter, Margaret A. Phillips

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This chapter discusses the design of mutant proteins, including mutagenesis, expression, and analysis. It focuses on the mutagenesis of substrate binding sites with the caveat that the optimization of enzyme activity may involve residues outside the active site. The use of sequence alignments, structure, and modeling is illustrated in the chapter from the work on substrate specificity of the serine proteases, trypsin and chymotrypsin. Trypsin cleaves at lysine and arginine P1 residues, whereas chymotrypsin prefers large hydrophobic residues in the P1 position. The main-chain structures of the two enzymes are virtually superimposable, and they are 40% identical in amino acid sequence. The differences in trypsin and chymotrypsin specificities most likely result from differences in the structure of their SI binding sites. The most straightforward approach to locate the substrate binding site is to determine the structure of enzyme–substrate or enzyme–inhibitor complexes. To a first approximation, the residues within 7 Å of the substrate/inhibitor are responsible for binding. Substrate binding sites can also be localized by chemical modification and mutagenesis experiments. The residues that confer substrate specificity can be further delineated when the enzyme is a member of a family of enzymes with different substrate specificities. The alignment of the primary sequences of these homologous proteins reveals conserved residues that may have important mechanistic or structural roles. Alignments also identify characteristic motifs associated with substrate specificities, and hence provide candidates for the determinants of specificity.

Original languageEnglish
Pages (from-to)671-687
Number of pages17
JournalMethods in enzymology
Issue numberC
Publication statusPublished - Jan 1991


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Hedstrom, L., Graf, L., Stewart, C. B., Rutter, W. J., & Phillips, M. A. (1991). Modulation of Enzyme Specificity by Site-Directed Mutagenesis. Methods in enzymology, 202(C), 671-687.