Structure-Based Design of an Intramolecular Proton Transfer Site in Murine Carbonic Anhydrase V†

Biochemistry ◽  
1996 ◽  
Vol 35 (36) ◽  
pp. 11605-11611 ◽  
Author(s):  
Robert W. Heck ◽  
P. Ann Boriack-Sjodin ◽  
Minzhang Qian ◽  
Chingkuang Tu ◽  
David W. Christianson ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Intramolecular Proton Transfer

Intramolecular Proton Transfer from Multiple Sites in Catalysis by Murine Carbonic Anhydrase V†

Biochemistry ◽  
1998 ◽  
Vol 37 (20) ◽  
pp. 7649-7655 ◽  
Author(s):  
J. Nicole Earnhardt ◽  
Minzhang Qian ◽  
Chingkuang Tu ◽  
Philip J. Laipis ◽  
David N. Silverman
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Intramolecular Proton Transfer

Intermolecular proton transfer in catalysis by carbonic anhydrase V

1999 ◽  
Vol 77 (5-6) ◽  
pp. 726-732 ◽  
Author(s):  
J Nicole Earnhardt ◽  
Chingkuang Tu ◽  
David N Silverman
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Active Site ◽  
Rate Constants ◽  
Intramolecular Proton Transfer ◽  
Proton Donor ◽  
Marcus Theory ◽  
Intermolecular Proton Transfer ◽  
Donor And Acceptor ◽  
Active Site Cavity

The dehydration of bicarbonate catalyzed by carbonic anhydrase is accompanied by the transfer of a proton from solution to the zinc-bound hydroxide. We have investigated the properties of proton transfer from donors in solution, mostly derivatives of imidazole and pyridine, to a truncated mutant of carbonic anhydrase V with replacements that render the active site cavity less sterically constrained, Tyr 64 →> Ala and Phe 65 →> Ala. Catalysis was measured by determining the rate of exchange of 18O between the CO2-HCO3- system and water, and rate constants for proton transfer were estimated as the rate-limiting step in the release of H218O from the enzyme to solution. Each proton donor enhanced catalytic activity in a saturable manner. The resulting rate constants for proton transfer when compared with the values of pKa of the donor and acceptor gave a Brønsted plot of high curvature. These data could also be described by Marcus theory which showed an intrinsic barrier for intermolecular proton transfer near 0.8 kcal/mol and a work term or thermodynamic contribution to the free energy of reaction near 10 kcal/mol. This low intrinsic kinetic barrier for proton transfer is very similar to nonenzymic bimolecular proton transfer between nitrogen and oxygen acids and bases in solution. However, the significant thermodynamic contribution suggests appreciable involvement of solvent and active-site organization prior to proton transfer. These Marcus parameters are very similar to those describing intramolecular proton transfer from His 64 in carbonic anhydrase, suggesting similarities in the intra- and intermolecular proton transfer processes.Key words: carbonic anhydrase, proton transfer, Marcus theory, carbon dioxide.

Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III

Biochemistry ◽  
1993 ◽  
Vol 32 (40) ◽  
pp. 10757-10762 ◽  
Author(s):  
David N. Silverman ◽  
Chingkuang Tu ◽  
Xian Chen ◽  
Susan M. Tanhauser ◽  
A. Jerry Kresge ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Intramolecular Proton Transfer ◽  
Carbonic Anhydrase Iii ◽  
Human Carbonic Anhydrase

The catalytic mechanism of carbonic anhydrase

1991 ◽  
Vol 69 (5) ◽  
pp. 1070-1078 ◽  
Author(s):  
David N. Silverman
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Catalytic Mechanism ◽  
Surrounding Medium ◽  
Bound Water ◽  
Intramolecular Proton Transfer ◽  
Carbonic Anhydrases ◽  
Diffusion Controlled ◽  
Active Site Cavity ◽  
Rate Limiting

Water as a ligand of the zinc in carbonic anhydrase has a pKa of 7 or less and the zinc-bound hydroxide is enhanced as a nucleophile for attack on CO2. The product of catalysis is HCO3− and a proton, and the catalytic pathway as determined for vertebrate isozymes I, II, and III occurs in two separate and distinct stages. The first stage includes the hydration of CO2 and ends with the release of HCO3− from its binding site as a ligand of the zinc; its position is replaced by a water molecule. This process is described by the ratio kcat/Km; for hydration catalyzed by isozyme II, the most efficient of the carbonic anhydrases, kcat/Km is close to diffusion controlled at 108 M−1∙s−1. The second stage is the regeneration of the zinc-bound hydroxide by protolysis of water and release of a proton to the surrounding medium. For carbonic anhydrase II, this proton transfer is rate determining for the maximal turnover number kcat of 106 s−1. Its pathway includes intramolecular proton transfer from the zinc-bound water to His64 in the active-site cavity followed by proton transfer to buffer in solution. For the least efficient of the carbonic anhydrases, isozyme III, kcat/Km near 3 × 105 M−1∙s−1 is not diffusion controlled; nevertheless, proton transfer from zinc-bound water to solution is still rate limiting for a maximal turnover of 104 s−1. Carbonic anhydrase isolated from spinach chloroplasts is quite similar to vertebrate isozyme II in catalytic properties, although it has been found to have almost no sequence homology with the vertebrate carbonic anhydrases. Key words: carbonic anhydrase, CO2, catalytic mechanism, proton transfer, bicarbonate.

scholarly journals Properties of Intramolecular Proton Transfer in Carbonic Anhydrase III

1998 ◽  
Vol 74 (6) ◽  
pp. 3182-3189 ◽  
Author(s):  
Chingkuang Tu ◽  
Minzhang Qian ◽  
J. Nicole Earnhardt ◽  
Philip J. Laipis ◽  
David N. Silverman
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Intramolecular Proton Transfer ◽  
Carbonic Anhydrase Iii
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