Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid)

Abstract

Carboxypeptidase A1 has been the subject of extensive research in the last 30 y and is one of the most widely studied zinc metalloenzymes. However, the three-dimensional structure of the human form of the enzyme is not yet available. This report describes the three-dimensional structure of human carboxypeptidase A1 (hCPA1) derived from crystals that belong to the tetragonal space group P4(3)2(1)2 and diffract to 1.6 angstroms resolution. A description of the ternary complex hCPA1-Zn2+-poly(acrylic acid) is included as a model of the interaction of mucoadhesive polymers with proteases in the gastrointestinal tract. The direct mode of interaction between poly(acrylic acid) and the active site of the target protease was confirmed by in vitro inhibition assays. The structure was further analyzed in silico through the optimal docking-area method. The characterization of binding sites on the surface of hCPA1 and a comparison with other available carboxypeptidase structures provided further insights into the formation of multiprotein complexes and the activation mechanisms of carboxypeptidase zymogens. The high-resolution structure of hCPA1 provides an excellent template for the modelling of physiologically relevant carboxypeptidases and could also contribute to the design of specific agents for biomedical purposes.

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

“…Kinetic constants were determined for CPA4 and also for human CPA1 and CPA2 to directly compare these related enzymes with the same substrates. CPA1 and CPA2 were obtained as recombinant proteins in the P. pastoris system, purified in their zymogen form, activated with trypsin, and further purified as described previously (32,33). k cat , K m , and k cat /K m values are shown in Table 1 and indicate that although all three enzymes have overlapping specificities, they differ markedly.…”

Crystal Structure of Novel Metallocarboxypeptidase Inhibitor from Marine Mollusk Nerita versicolor in Complex with Human Carboxypeptidase A4

“…The experiments were performed at 37°C and pH 7.5 by varying the inhibitor concentration in each assay with a fixed concentration of enzyme and substrate (0.1 mM N-(4-methoxyphenylazoformyl)phenylalanine) and preincubation time. K i values were determined for bCPA1 (Sigma-Aldrich) and for hCPA1, hCPA2, and hCPA4, which were produced by recombinant expression by our group (11,27,28).…”

The X‐Ray Structure of Carboxypeptidase A Inhibited by a Thiirane Mechanism‐Based Inhibitor

“…Tyr-248 has been observed in two conformational states in the several structures available for CPA. One brings it to a hydrogen bonding distance of the bound peptide substrate (the ‘closed’ position) and the other is away from it (the ‘open’ position) (38,39). In the X-ray structure of the inhibited complex, the Tyr-248 phenol group moves from the surface closer to the active site cleft to make a strong hydrogen bond to the carboxylate group of the inhibitor (the bond distance is 2.59 Å; the ‘closed’ position).…”