Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L-glutamic acid: reversed-polarity mutants

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity fo r hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared b y site-directed mutagenesis of the human gene and ex pressed in the peripla sm of Escherichia coli, By changing residues in the lining of the S1' pocke t of the enzyme, it was possible to reverse the substrate specificity to gi ve variants able to hydrolyse prior to C-terminal acidic amino acid residue s instead of the normal C-terminal basic residues. This was achieved by mut ating Asp253 at the base of the S1' specificity pocket, which normally inte racts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity wa s improved significantly with further mutations at residue 251, The [G251T, D253K]HCPB double mutant was 100 times more active against hippuryl-L-gluta mic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. T riple mutants, containing additional changes at Ala238, had improved activi ty against hipp-Glu substrate when position 251 was Asn, These reversed-pol arity mutants of a human enzyme have the potential to be used in antibody-d irected enzyme prodrug therapy of cancer.