Modified carboxypeptidase

Abstract

A method for transamidating a peptide substrate having a P 1 amino acid residue with a positively charged side chain. According to the invention, carboxypeptidase Y is modified to substitute at least one amino acid having a negatively charged side chain in an S 1 subsite. Additionally, the modified carboxypeptidase Y can include substituted amino acid residues in an S 1 ′, S 2 and/or S 3 subsite to accommodate a specific peptide substrate.

We claim:  
     
         1 . A method for transamidating a peptide substrate having a P 1  amino acid residue with a positively charged side chain, the method comprising: 
 reacting the peptide substrate with a nucleophile in the presence of a modified carboxypeptidase Y, wherein the modified carboxypeptidase comprises at least one substitution in an S 1  subsite with an amino acid having a negatively charged side chain.    
     
     
         2 . The method of    claim 1   , wherein the modified carboxypeptidase comprises at least one substitution at amino acid residue L178, W312, N241, or L245.  
     
     
         3 . The method of    claim 2   , wherein at least one of L178, W312, N241, or L245 is replaced by aspartic acid or glutamic acid.  
     
     
         4 . The method of    claim 1   , wherein the nucleophile comprises an amino acid ester or amino acid amide.  
     
     
         5 . The method of    claim 4   , wherein the nucleophile comprises Leu-OH, Leu-OR, or Leu-NRR′.  
     
     
         6 . The method of    claim 1   , wherein the modified carboxypeptidase further comprises at least one substituted amino acid residue in an S 1 , S 2 , or S 3  subsite.  
     
     
         7 . The method of    claim 1   , wherein the modified carboxypeptidase further comprises at least one substitution in an S 1 ′ subsite selected from the group consisting of N51Q, T60W, T60Y, T60A M398L, M398G M398A, M398V, M3981, M398F, M398Y, M398C, M398N, and M398E.  
     
     
         8 . The method of    claim 7   , wherein the modified carboxypeptidase further comprises two or more amino acid substitutions in the S 1 ′ subsite.  
     
     
         9 . The method of    claim 8   , wherein the two or more substitutions in the S 1 ′ subsite include at least one substitution at position T60 and at least one substitution at position M398.  
     
     
         10 . The method of    claim 9   , wherein the substitution at position T60 is T60F, T60L, T60Y, or T60D.  
     
     
         11 . The method of    claim 9   , wherein the substitution at position M398 is M398F, M398L, or M398Y.  
     
     
         12 . The method of    claim 1   , wherein the modified carboxypeptidase further comprises at least one substitution of an amino acid in an S 2  subsite.  
     
     
         13 . The method of    claim 12   , wherein the substitution in the S 2  subsite is in at least one of positions E296, S 297,  or N303.  
     
     
         14 . The method of    claim 12   , wherein the substitution in the S 2  subsite is E296F, S297A, N303A, or N303F.  
     
     
         15 . The method of    claim 12   , wherein the peptide substrate includes an Ala residue at P 2 .  
     
     
         16 . The method of claim l, wherein the modified carboxypeptidase Y further comprises at least one secondary substitution of an amino acid residue capable of interacting with a peptide substrate P 3  residue having a positively charged side chain.  
     
     
         17 . The method of    claim 1   , wherein the modified carboxypeptidase comprises at least one secondary substitution of an amino acid residue capable of interacting with a peptide substrate P 3  residue wherein the secondary substitution introduces an amino acid having a negatively charged side chain.  
     
     
         18 . The method of    claim 16   , wherein the secondary substitution is in at least one of positions N241, N242, L245, or A246.  
     
     
         19 . The method of    claim 1   , wherein reacting is at about pH 6.3 to about pH 6.7.  
     
     
         20 . A modified carboxypeptidase Y, comprising a substituted amino acid in at least one of positions N241 or L245 wherein the substituted amino acid has a negatively charged side chain.  
     
     
         21 . The modified carboxypeptidase of    claim 20   , further comprising at least one substituted amino acid residue in an S 1 ′, S 2  or S 3  subsite.  
     
     
         22 . The modified carboxypeptidase of    claim 20   , further comprising a substituted amino acid residue in at least one of positions L 178 or W312.  
     
     
         23 . The modified carboxypeptidase of    claim 20   , further comprising substitutions L178S and M398L.