The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over.
The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.
Deiteren
Kathleen
K
Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. kathleen.deiteren@ua.ac.be
Surpateanu
Georgiana
G
Gilany
Kambiz
K
Willemse
Johan L
JL
Hendriks
Dirk F
DF
Augustyns
Koen
K
Laroche
Yves
Y
Scharpé
Simon
S
Lambeir
Anne-Marie
AM
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D058851:GPI-Linked Proteins; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D058851:GPI-Linked Proteins; D011994:Recombinant Proteins; C058479:carboxypeptidase M; D008666:Metalloendopeptidases