Jf. Krebs et al., KINETIC AND SPECTROSCOPIC STUDIES OF HYDROPHILIC AMINO-ACID SUBSTITUTIONS IN THE HYDROPHOBIC POCKET OF HUMAN CARBONIC ANHYDRASE-II, Biochemistry, 32(17), 1993, pp. 4496-4505
The functional importance and structural determinants of a conserved h
ydrophobic pocket in human carbonic anhydrase II (CA II) were probed b
y preparing and characterizing 13 amino acid substitutions at Leu-198,
situated at the mouth of the pocket. The pH dependence of the esteras
e activity reveals that activity decreases (up to 120-fold) as the ami
no acid size and charge at position 198 are varied while the pK(a) of
the zinc-bound water molecule increases (up to 1 pH unit). Intriguingl
y, the pH dependence of the Leu-198-Glu substitution is parabolic (pK(
a)s almost-equal-to 6 and 9), consistent with introduction of a genera
l base-catalyzed mechanism. Kinetic characterization of CO2/HCO3- inte
rconversion catalyzed by four variants (Leu-198-Ala, His, Arg, and Glu
) reveals that increasing the size of the hydrophobic pocket (Ala) doe
s not compromise catalysis (almost-equal-to 3-fold decrease); however,
substitution of charged (Arg and Glu) and larger (His) amino acids de
creases k(cat)/K(M) for CO2 hydration substantially (17-fold, 19-fold,
and 10-fold, respectively) but not completely. log k(cat)/K(M) for CO
2 hydration, HCO3- dehydration, and p-nitrophenyl acetate hydrolysis c
orrelates with the hydrophobicity of the residue at 198, likely reflec
ting desolvation or electrostatic destabilization of the ground state.
The X-ray crystal structures of the Leu-198-His, Glu, and Arg variant
s (Nair & Christianson, 1993) indicate that the His and Glu side chain
s are accommodated by minor structural reorganization leading to a wid
er mouth for the hydrophobic pocket while the Arg side chain blocks th
e pocket. Infrared spectroscopy of CO2 bound to either wild-type CA II
or the Leu-198-->Arg variant indicates that the Arg substitution both
decreases the affinity and alters the position of CO2 binding, sugges
ting that the hydrophobic pocket forms the CO2 binding site in CA II.
Finally, a 1.5-fold increase (Leu-198-->Ala) and 12-fold decrease (Leu
-198-->Arg) in k(cat) for CO2 hydration, indicative of the rate consta
nt for intramolecular proton transfer from zinc-bound water to His-64,
are likely mediated by changes in the active site solvent structure.