Crystal Structures of Complexes with Cobalt-Reconstituted Human Arginase I

The binuclear manganese metalloenzyme human arginase I (HAI) is a potential protein drug for cancer chemotherapy, in that it is capable of depleting extracellular L-Arg levels in the microenvironment of tumor cells that require this nutrient to thrive. Substitution of the native Mn-2(2+) cluster with a Co-2(2+) duster in the active site yields an enzyme with enhanced catalytic activity at physiological pH (similar to 7.4) that could serve as an improved protein drug for L-Arg depletion therapy [Stone, E. M., Glazer, E. S., Chantranupong, L., Cherukuri, P., Breece, R. M., Tierney, D. L., Curley, S. A., Iverson, B. L., and Georgiou, G. (2010) ACS Chem. Biol. S, 333-342]. A different catalytic mechanism is proposed for Co-2(2+)-HAI compared with that of Mn-2(2+)-HAI, including an unusual N epsilon-Co2+ coordination mode, to rationalize the lower K-M value of L-Arg and the lower K-i value of L-Orn. However, we now report that no unusual metal coordination modes are observed in the cobalt-reconstituted enzyme. The X-ray crystal structures of unliganded Co-2(2+)-HAI determined at 2.10 angstrom resolution(pH 7.0) and 1.97 angstrom resolution (pH 8.5), as well as the structures of Co-2(2+)-HA1 complexed with the reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH, pH 7.0) and the catalytic product L-Orn (pH 7.0) determined at 1.85 and 1.50 angstrom resolution, respectively, are essentially identical to the corresponding structures of Mn-2(2+)-HAI. Therefore, in the absence of significant structural differences between Co-2(2+)-HAI and Mn-2(2+)-HAI, we suggest that a higher concentration of metal-bridging hydroxide ion at physiological pH for Co-2(2+)-HAI, a consequence of the lower pK(a) of a Co2+-bound water molecule compared with a Mn2+-bound water molecule, strengthens electrostatic interactions with cationic amino acids and accounts for enhanced affinity as reflected in the lower K-M value of L-Arg and the lower K-i value of L-Orn.