Comparative substrate specificity study of carboxypeptidase U (TAFIa) and carboxypeptidase N: development of highly selective CPU substrates as useful tools for assay development.

Background: Measurement of procarboxypeptidase U (TAFI) in plasma by activity-based assays is complicated by the presence of plasma carboxypeptidase N (CPN). Accurate blank measurements, correcting for this interfering CPN activity, should therefore be performed. A selective CPU substrate will make proCPU determination much less time-consuming.

Methods: We searched for selective and sensitive CPU substrates by kinetic screening of different Bz-Xaa-Arg (Xaa=a naturally occurring amino acid) substrates using a novel kinetic assay.

Results: The presence of an aromatic amino acid (Phe, Tyr, Trp) resulted in a fairly high selectivity for CPU which was most pronounced with Bz-Trp-Arg showing a 56-fold higher k(cat)/K(m) value for CPU compared to CPN. Next we performed chemical modifications on the structure of those aromatic amino acids. This approach resulted in a fully selective CPU substrate with a 2.5-fold increase in k(cat) value compared to the commonly used Hip-Arg (Bz-Gly-Arg).

Discussion: We demonstrated significant differences in substrate specificity between CPU and CPN that were previously not fully appreciated. The selective CPU substrate presented in this paper will allow straightforward determination of proCPU in plasma in the future.