The catalytic mechanism of glucose 6-phosphate dehydrogenases:: Assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme
The catalytic mechanism of glucose 6-phosphate dehydrogenases:: Assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme
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DOI：
10.1021/bi0255219
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发表时间：
2002-06-04
期刊：
影响因子：
2.9
通讯作者：
Levy, HR
中科院分区：
文献类型：
--
作者：
Cosgrove, MS;Loh, SN;Levy, HR
The chemical shifts of the C-is an element of1 and C-delta2 protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing H-1 and C-13 spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 H-1(is an element of1) resonance was obtained from comparing C-13-H-1 heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C I histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of k(cat) [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that H-1 NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.
