The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178.
A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Olesen
K
K
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Mortensen
U H
UH
Aasmul-Olsen
S
S
Kielland-Brandt
M C
MC
Remington
S J
SJ
Breddam
K
K
D000595:Amino Acid Sequence; D000599:Amino Acids, Diamino; D002268:Carboxypeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D043402:Cathepsin A; D004926:Escherichia coli; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000599:Amino Acids, Diamino; D009842:Oligopeptides; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; D007930:Leucine