Structural and Kinetic Characterization of Active-Site Histidine as a Proton Shuttle in Catalysis by Human Carbonic Anhydrase II

详细信息    查看全文

• 作者：Zoë ; Fisher ; Jose A. Hernandez Prada ; Chingkuang Tu ; David Duda ; Craig Yoshioka ; Haiqian An ; Lakshmanan Govindasamy ; David N. Silverman ; and Robert McKenna
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：February 1, 2005
• 年：2005
• 卷：44
• 期：4
• 页码：1097 - 1105
• 全文大小：356K
• 年卷期：v.44,no.4(February 1, 2005)
• ISSN：1520-4995

文摘

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by humancarbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solventmolecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH onproton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and twodouble mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chainsextending into the active-site cavity with distances from the zinc approximately equivalent to that ofHis64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of ¹⁸O betweenCO₂ and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10⁵ s^-1was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at arate near 10³ s^-1 was observed for His62. The observation, in the crystal structures, of a completedhydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does notappear to be required for efficient proton transfer. The data suggest that the number of intervening watermolecules between the donor and acceptor supporting efficient proton transfer in HCA II is important,and furthermore suggest that a water bridge consisting of two intervening water molecules is consistentwith efficient proton transfer.