Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-arginine

Abstract

Porcine pancreatic carboxypeptidase B (EC 3.4.23.6) was complexed with a stable transition-state analogue, N-sulfamoyl-l-arginine, in which an S atom imitates the sp 3 -hybridized carbon in the scissile-bond surrogate. Crystals were grown in a form belonging to the same space group, P4 1 2 1 2, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 Å . The molecule was refined and the positions of non-H atoms of the inhibitor and water molecules were defined using difference Fourier maps. The enzyme-inhibitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation between the complexed and uncomplexed forms of carboxypeptidase B are shown. The inhibitor is bound in a curved conformation in the active-site cleft, and the sulfamide group is bound to the Zn ion in an asymmetric bidentate fashion. The complex is stabilized by hydrogen bonds between the N1/N2 guanidine group of the inhibitor and the Asp255 carboxyl of the enzyme. The side-chain CH 2 groups of the inhibitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may bind to carboxypeptidase B and therefore provides an insight into the structural basis of carboxypeptidase B selectivity, which is useful for the rational design of a carboxypeptidase with improved selectivity for industrial recombinant proinsulin processing.

The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

“…The spatial structures of these complexes generally correspond to the structures of previously obtained complexes of N-sulfamoyl phenylalanine (CPA+SPhe) [39] and N-sulfamoyl arginine (CPT+SArg, CPB+SArg) [40, 11] with carboxypeptidases A, B and T. The tetrahedral sulfamoyl group, that mimics the tetrahedral sp 3 -hybridized carbon atom of the convertible bond, interacts with the residues of the catalytic site of CPT, including the zinc ion coordinated by the side chain functional groups of Glu72, His204, His69, the catalytic residue Glu277, which activates the attacking water molecule, and Arg129, which polarizes the carbonyl of the bond to be cleaved. The sulfamoyl group of the ligand is located asymmetrically with respect to the Zn 2+ ion.…”

“…A different situation was observed in case of CPB, a homologous enzyme with narrow substrate specificity. When moving from the SPhe complex [41] to the SArg complex [40], the Zn-S distance does not change (Fig 3). However, due to the rotation of the ligand around its axis, the side chain carboxyl of Glu270 is shifted by 0.28 Å, the C-terminal oxygen atom by 0.26 Å, and the phenol hydroxyl of Tyr248 by 0.32 Å.…”

Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

“…We obtained an X-ray cocrystal structure of the complex. On the basis of this and a report by Ryu et al where a sulfamide acts as zinc-binder in transition state analogue inhibitors of carboxypeptidase A, we speculated that we could expand the chemical space by replacing the urea zinc binder in our previously reported TAFIa inhibitors with a sulfamide moiety . To explore this unusual zinc binding motif, we prepared the direct sulfamide analogue of our previous lead structure (Figure ) by the general synthesis route described in Scheme .…”

Microgravity Crystal Formation

“…Molecular weight was used as a surrogate for molecular complexity, and the implication was that complex systems show the same benefits of crystallization as simple systems. In fact, several proteins had their first reports of successful crystallization in a microgravity environment: carboxypeptidase T [36]; carboxypeptidate B complex with N-sulfamoyl-L-arginine [37]; T3Ri insulin hexamer [38]; phosphoribosyl pyrophosphate synthetase from E. coli [39]; E. coli purine nucleoside phosphorylase in complex with 7deazahypoxanthin [40]; and recombinant phosphoribosylpyrophosphate synthetase-2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions [41].…”