Unknown

Dataset Information

0

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II.


ABSTRACT: The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹?O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO?, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.

SUBMITTER: Mikulski R 

PROVIDER: S-EPMC3044917 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

altmetric image

Publications

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II.

Mikulski Rose R   Avvaru Balendu Sankara BS   Tu Chingkuang C   Case Nicolette N   McKenna Robert R   Silverman David N DN  

Archives of biochemistry and biophysics 20101209 2


The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had n  ...[more]

Similar Datasets

| S-EPMC3223279 | biostudies-literature
| S-EPMC2925223 | biostudies-literature
| S-EPMC5755581 | biostudies-literature
| S-EPMC2576554 | biostudies-literature
| S-EPMC2967421 | biostudies-literature
| S-EPMC2865367 | biostudies-literature
| S-EPMC2810610 | biostudies-literature
| S-EPMC2736349 | biostudies-literature
| S-EPMC3378272 | biostudies-literature
| S-EPMC2150928 | biostudies-literature