Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase.
We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.
Tu
Chingkuang
C
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610-0267, USA.
Qian
Minzhang
M
An
Haiqian
H
Wadhwa
Nina R
NR
Duda
David
D
Yoshioka
Craig
C
Pathak
Yashash
Y
McKenna
Robert
R
Laipis
Philip J
PJ
Silverman
David N
DN
D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism*; D024403:Carbonic Anhydrase III / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction; D015032:Zinc / Q000737:chemistry
D007527:Isoenzymes; D011522:Protons; D014867:Water; D002245:Carbon Dioxide; D006639:Histidine; D024402:Carbonic Anhydrase II; D024403:Carbonic Anhydrase III; D015032:Zinc; D010100:Oxygen