Krebs J F, Rana F, Dluhy R A, Fierke C A
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Biochemistry. 1993 May 4;32(17):4496-505. doi: 10.1021/bi00068a004.
The functional importance and structural determinants of a conserved hydrophobic pocket in human carbonic anhydrase II (CA II) were probed by preparing and characterizing 13 amino acid substitutions at Leu-198, situated at the mouth of the pocket. The pH dependence of the esterase activity reveals that activity decreases (up to 120-fold) as the amino acid size and charge at position 198 are varied while the pKa of the zinc-bound water molecule increases (up to 1 pH unit). Intriguingly, the pH dependence of the Leu-198-->Glu substitution is parabolic (pKas approximately 6 and 9), consistent with introduction of a general base-catalyzed mechanism. Kinetic characterization of CO2/HCO3- interconversion catalyzed by four variants (Leu-198-->Ala, His, Arg, and Glu) reveals that increasing the size of the hydrophobic pocket (Ala) does not compromise catalysis (approximately 3-fold decrease); however, substitution of charged (Arg and Glu) and larger (His) amino acids decreases kcat/KM for CO2 hydration substantially (17-fold, 19-fold, and 10-fold, respectively) but not completely. log kcat/KM for CO2 hydration, HCO3- dehydration, and p-nitrophenyl acetate hydrolysis correlates with the hydrophobicity of the residue at 198, likely reflecting desolvation or electrostatic destabilization of the ground state. The X-ray crystal structures of the Leu-198-->His, Glu, and Arg variants (Nair & Christianson, 1993) indicate that the His and Glu side chains are accommodated by minor structural reorganization leading to a wider mouth for the hydrophobic pocket while the Arg side chain blocks the 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, suggesting 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 kcat for CO2 hydration, indicative of the rate constant for intramolecular proton transfer from zinc-bound water to His-64, are likely mediated by changes in the active site solvent structure.
通过在位于疏水口袋开口处的亮氨酸-198位点制备并表征13种氨基酸取代，探究了人碳酸酐酶II（CA II）中保守疏水口袋的功能重要性和结构决定因素。酯酶活性的pH依赖性表明，随着198位氨基酸大小和电荷的变化，活性降低（高达120倍），而锌结合水分子的pKa增加（高达1个pH单位）。有趣的是，亮氨酸-198突变为谷氨酸的pH依赖性呈抛物线形（pKa约为6和9），这与引入一般碱催化机制一致。对四种变体（亮氨酸-198突变为丙氨酸、组氨酸、精氨酸和谷氨酸）催化的CO2/HCO3-相互转化的动力学表征表明，增大疏水口袋的大小（丙氨酸）不会损害催化作用（约降低3倍）；然而，带电（精氨酸和谷氨酸）和较大（组氨酸）氨基酸的取代会使CO2水合的kcat/KM大幅降低（分别为17倍、19倍和10倍），但并非完全消除。CO2水合、HCO3-脱水和对硝基苯乙酸水解的log kcat/KM与198位残基的疏水性相关，这可能反映了基态的去溶剂化或静电不稳定。亮氨酸-198突变为组氨酸、谷氨酸和精氨酸变体的X射线晶体结构（Nair和Christianson，1993）表明，组氨酸和谷氨酸侧链通过轻微的结构重排得以容纳，导致疏水口袋开口变宽，而精氨酸侧链则阻塞了口袋。与野生型CA II或亮氨酸-198突变为精氨酸变体结合的CO2的红外光谱表明，精氨酸取代既降低了亲和力，又改变了CO2结合的位置，这表明疏水口袋构成了CA II中的CO2结合位点。最后，CO2水合的kcat增加1.5倍（亮氨酸-198突变为丙氨酸）和降低12倍（亮氨酸-198突变为精氨酸），这表明分子内质子从锌结合水转移到组氨酸-64的速率常数，可能是由活性位点溶剂结构的变化介导的。
