Imidazole C-2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

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• 作者：Naoka Hayashi ; Hiroki Kuyama ; Chihiro Nakajima ; Kazuki Kawahara ; Masaru Miyagi ; Osamu Nishimura ; Hisayuki Matsuo ; Takashi Nakazawa
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：March 25, 2014
• 年：2014
• 卷：53
• 期：11
• 页码：1818-1826
• 全文大小：442K
• 年卷期：v.53,no.11(March 25, 2014)
• ISSN：1520-4995

文摘

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine C^蔚1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second-order with respect to the concentrations of the imidazolium cation and OD^{鈥?/sup> in D₂O. The second-order rate constant (k₂) of this reaction was calculated from the pH dependency of the pseudo-first-order rate constant (k_蠁) obtained from the change in average mass [螖M_r (0 鈮?螖M_r < 1)] of a peptide fragment containing a defined histidine residue at incubation time (t) such that k_蠁 = 鈭抂ln(1 鈥?螖M_r)]/t. We preferred using k₂ rather than k_蠁 because k₂max (maximal value of k₂) was empirically related to pK_a as illustrated with a Br酶nsted plot [log k₂max = 鈭?.7pK_a + 伪 (伪 is an arbitrary constant)], so that we could analyze the effect of structure on the H/D exchange rate in terms of log(k₂max/k₂) representing the deviation of k₂ from k₂max. In the catalytic site of bovine ribonuclease A, His12 showed a change in log(k₂max/k₂) much larger than that of His119 upon binding with cytidine 3鈥?monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, we need to consider the hydrogen bonds of the imidazole group with nondissociable groups to interpret an extremely slow H/D exchange rate of His48 in a partially solvent-exposed situation.}