Catalytic and Inhibitor‐Binding Properties of Some Active‐Site Mutants of Human Carbonic Anhydrase I

Abstract

Three isozyme-specific residues in the active site of human carbonic anhydrase I, Va162, His67, and His200, have been changed by site-directed mutagenesis to their counterparts in human carbonic anhydrase 11, Asn62, Asn67, and Thr200. A double mutant, containing Asn62 and Asn67, and a triple mutant, containing all three alterations, were also produced. The rates of CO, hydration and ester hydrolysis catalyzed by these mutants, the inhibition of these enzymes by the anions, SCN-, and I-, and the binding of the sulfonamide inhibitors, dansylamide and MK-417 (a thienothiopyran-2-sulfonamide) have been measured. The results suggest that the effect of His200 in isozyme I is to prolong the lifetime of the enzyme-bicarbonate complex and to increase the pK, of the catalytic group, a zinc-coordinated water molecule. For isozyme I, Val62 and His67 might interfere with the function of a proton 'shuttle' group in the active site, thus maintaining the buffer specificity of a compulsory proton-transfer step. The single mutations have small effects on anion binding. Only the triple mutant has anion-binding properties resembling those of isozyme 11. All mutants show altered sulfonamide-binding properties. In particular, the binding specificity is affected. While wild-type isozyme I binds dansylamide SO times more strongly than MK-417, the triple mutant shows a reversed selectivity and binds MK-417 nearly SO times more strongly than dansylamide.

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…Technique -Inhibition FTSA, ITC CA I (Morkūnaitė et al, 2015) 8.1 (Coleman, 1967) 8.4 ± 0.2 8.1 −47.9 −48.2 −41.0 −38.5 6.9 9.7 6.9 (Innocenti et al, 2009) 7.1 (Engstrand et al, 1995) 7.3 (Whitney and Brandt, 1976;Khalifah, 1977;Khalifah et al, 1977) CA II (Morkūnaitė et al, 2015) 6.9 (Duda et al, 2001;Mikulski et al, 2011a) 7.1 6.9 −40.5 −41.1 −26.0 −23.5 14.5 17.6 7.1 (Innocenti et al, 2009) 6.8 (Tu et al, 2002) CA III ∼5 (Tu et al, 2002) 6.6 6.5 −37.7 −38.6 ND ND ND ND…”

Structural Features that Govern Enzymatic Activity in Carbonic Anhydrase from a Low-Temperature Adapted Fish, Chionodraco hamatus

“…For instance, His 64 is the catalytic proton shuttle in CAII (k cat ¼ 10 6 s À1 ) (11,12) and the tautomerization of this residue can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time-or energy-consuming processes (13). The side chain of His 200 is the best candidate for a proton shuttle in CAI (k cat ¼ 2 3 10 5 s À1 ) (14) due to its proximity to the zinc ion and to the observed pKa value (15). In contrast, it seems that no proton shuttle is present in the active site of CAIII (k cat ¼ 1 3 10 4 s À1 ), in which the product proton is probably transferred directly to the bulk solvent (16).…”