1urt
From Proteopedia
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==Overview== | ==Overview== | ||
| - | Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the, hydration of CO2 to produce bicarbonate and a proton. The catalytic, properties of wild-type murine CA V suggest the presence of a proton, shuttle residue having pKa = 9.2, the role of which is to transfer a, proton from zinc-bound water to solution in the hydration direction to, regenerate the zinc hydroxide form of the enzyme. Two likely candidates, for shuttle residues are the tyrosines at positions 64 and 131 in the, active site cavity. The crystal structure of wild-type carbonic anhydrase, V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an, orientation pointing away from the zinc by Phe 65, although Tyr 131 is, oriented toward the ... | + | Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the, hydration of CO2 to produce bicarbonate and a proton. The catalytic, properties of wild-type murine CA V suggest the presence of a proton, shuttle residue having pKa = 9.2, the role of which is to transfer a, proton from zinc-bound water to solution in the hydration direction to, regenerate the zinc hydroxide form of the enzyme. Two likely candidates, for shuttle residues are the tyrosines at positions 64 and 131 in the, active site cavity. The crystal structure of wild-type carbonic anhydrase, V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an, orientation pointing away from the zinc by Phe 65, although Tyr 131 is, oriented toward the zinc. We have prepared mutants of murine CA V, replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the, proton shuttle mechanism using stopped-flow spectrophotometry and the, depletion of 18O from CO2 measured by mass spectrometry. Experiments with, both single and double mutations showed that neither position 64 nor, position 131 was a prominent site for proton transfer. However, a double, mutant of CA V containing the two replacements, Tyr 64-->His and Phe, 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of, 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to, the altered catalytic properties, the crystal structure of the His 64/Ala, 65 double mutant strongly suggested proton transfer by His 64 after, removal of the steric hindrance of Phe 65. This is the first, structure-based design of an efficient proton transfer site in an enzyme. |
==About this Structure== | ==About this Structure== | ||
| - | 1URT is a | + | 1URT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus] with ZN as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Carbonate_dehydratase Carbonate dehydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.1 4.2.1.1] Structure known Active Site: ZIN. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1URT OCA]. |
==Reference== | ==Reference== | ||
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[[Category: zinc]] | [[Category: zinc]] | ||
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 14:42:07 2007'' |
Revision as of 12:36, 5 November 2007
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MURINE CARBONIC ANHYDRASE V
Overview
Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the, hydration of CO2 to produce bicarbonate and a proton. The catalytic, properties of wild-type murine CA V suggest the presence of a proton, shuttle residue having pKa = 9.2, the role of which is to transfer a, proton from zinc-bound water to solution in the hydration direction to, regenerate the zinc hydroxide form of the enzyme. Two likely candidates, for shuttle residues are the tyrosines at positions 64 and 131 in the, active site cavity. The crystal structure of wild-type carbonic anhydrase, V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an, orientation pointing away from the zinc by Phe 65, although Tyr 131 is, oriented toward the zinc. We have prepared mutants of murine CA V, replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the, proton shuttle mechanism using stopped-flow spectrophotometry and the, depletion of 18O from CO2 measured by mass spectrometry. Experiments with, both single and double mutations showed that neither position 64 nor, position 131 was a prominent site for proton transfer. However, a double, mutant of CA V containing the two replacements, Tyr 64-->His and Phe, 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of, 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to, the altered catalytic properties, the crystal structure of the His 64/Ala, 65 double mutant strongly suggested proton transfer by His 64 after, removal of the steric hindrance of Phe 65. This is the first, structure-based design of an efficient proton transfer site in an enzyme.
About this Structure
1URT is a Single protein structure of sequence from Mus musculus with ZN as ligand. Active as Carbonate dehydratase, with EC number 4.2.1.1 Structure known Active Site: ZIN. Full crystallographic information is available from OCA.
Reference
Structure-based design of an intramolecular proton transfer site in murine carbonic anhydrase V., Heck RW, Boriack-Sjodin PA, Qian M, Tu C, Christianson DW, Laipis PJ, Silverman DN, Biochemistry. 1996 Sep 10;35(36):11605-11. PMID:8794740
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