Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.
The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.
Shimahara
Hideto
H
Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1211; Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871; Institute for Protein Research, Osaka University, Suita, Osaka 565-0871; Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.
Yoshida
Takuya
T
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871.
Shibata
Yasutaka
Y
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871.
Shimizu
Masato
M
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871.
Kyogoku
Yoshimasa
Y
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871.
Sakiyama
Fumio
F
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871.
Nakazawa
Takashi
T
Department of Chemistry, Nara Women's University, Nara 630-8506.
Tate
Shin-Ichi
SI
Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1211.
Ohki
Shin-Ya
SY
Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1211.
Kato
Takeshi
T
Medical School Osaka University, Suita, Osaka 565-0871.
Moriyama
Hozumi
H
Medical School Osaka University, Suita, Osaka 565-0871.
Kishida
Ken-Ichi
KI
Medical School Osaka University, Suita, Osaka 565-0871.
Tano
Yasuo
Y
Medical School Osaka University, Suita, Osaka 565-0871.
Ohkubo
Tadayasu
T
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871.
Kobayashi
Yuji
Y
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871; Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan. Electronic address: kobayasi@gly.oups.ac.jp.
D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002384:Catalysis; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D011522:Protons; D013816:Thermodynamics
D011522:Protons; D006639:Histidine; D002256:Carbonic Anhydrases