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Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.

ABSTRACT: The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.

SUBMITTER: Maupin CM

PROVIDER: S-EPMC2739621 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.

Maupin C Mark CM Zheng Jiayin J Tu Chingkuang C McKenna Robert R Silverman David N DN Voth Gregory A GA

Biochemistry 20090801 33

The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disord ...[more]

PMID: 19634894

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Project description:Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

Dataset Information

Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.

Publications

Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.

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