Pitson S M, Mendz G L, Srinivasan S, Hazell S L
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
Eur J Biochem. 1999 Feb;260(1):258-67. doi: 10.1046/j.1432-1327.1999.00153.x.
The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
利用[1H]-和[13C]-核磁共振光谱法及分光光度法，对微需氧人类病原体幽门螺杆菌三羧酸循环的组成和特性进行了原位及细胞提取物研究。核磁共振光谱分析能够在幽门螺杆菌的原位研究中对一些酶活性进行高度特异性测量，而这在以前使用分光光度法是无法实现的，从而首次准确描绘了该细菌完整的三羧酸循环。描述了幽门螺杆菌中柠檬酸合酶、乌头酸酶、异柠檬酸脱氢酶、α-酮戊二酸氧化酶、延胡索酸还原酶、延胡索酸酶、苹果酸脱氢酶和苹果酸合酶活性的存在、细胞定位及动力学参数。还显示了该循环中其他酶活性的缺失，包括α-酮戊二酸脱氢酶、琥珀酰辅酶A合成酶和琥珀酸脱氢酶。幽门螺杆菌的三羧酸循环似乎是一种非循环的分支途径，具有厌氧代谢的特征，在还原性二羧酸分支中导向琥珀酸的产生，在氧化性三羧酸分支中导向α-酮戊二酸的产生。两个分支通过α-酮戊二酸氧化酶活性的存在在代谢上相互联系。在所采用的生长条件下，由于缺乏异柠檬酸裂解酶活性，幽门螺杆菌不具备有效的乙醛酸旁路；也没有γ-氨基丁酸分流途径，因为缺乏γ-氨基丁酸转氨酶和琥珀酸半醛脱氢酶活性。通过将幽门螺杆菌三羧酸循环酶的氨基酸序列与其他研究更广泛的酶的序列进行比较，讨论了这些酶的催化和调节特性。
