Mendz G L, Hazell S L, Burns B P
School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.
Arch Biochem Biophys. 1994 Aug 1;312(2):349-56. doi: 10.1006/abbi.1994.1319.
Evidence for the presence of the enzymes of the Entner-Doudoroff pathway in Helicobacter pylori was obtained using 1H and 31P nuclear magnetic resonance spectroscopy. Bacterial lysates generated 6-phosphogluconate and NADH or NADPH in incubations with glucose-6-phosphate and NAD+ or NADP+, indicating the presence of glucose-6-phosphate dehydrogenase activities. Formation of pyruvate was observed in time courses of incubations of bacterial lysates with 6-phosphogluconate as the only substrate, suggesting the presence of 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase activities. The existence of these enzymes and of triose phosphate isomerase was confirmed by observing the appearance of dihydroxyacetone phosphate in time courses of bacterial lysates incubated with 6-phosphogluconate. Aldolase activity was measured by the production of pyruvate and dihydroxyacetone phosphate in lysates incubated with 2-keto-3-deoxy-6-phosphogluconate as the sole substrate. Dehydrogenase, dehydratase and aldolase activities were observed in several bacterial strains including wild types from fresh isolates. Kinetic parameters were measured for the three activities. The cellular location of the enzymes was investigated by comparing the activities measured in the pellet and supernatant fractions obtained by centrifugation of lysate suspensions. The concentration of compounds causing 50% inhibition of enzyme activity was determined from dose-response curves. The data suggested the presence of two glucose-6-phosphate dehydrogenases linked to NAD+ and NADP+ activities. Using inhibitors differences between the H. pylori and mammalian KDPG aldolases were detected. The presence of these enzyme activities in H. pylori provided evidence for the existence of the Entner-Douderoff pathway in the bacterium.
利用氢核磁共振光谱和磷核磁共振光谱，获得了幽门螺杆菌中存在恩特纳-杜德洛夫途径酶的证据。在细菌裂解物与6-磷酸葡萄糖和NAD⁺或NADP⁺的孵育过程中，产生了6-磷酸葡萄糖酸和NADH或NADPH，这表明存在6-磷酸葡萄糖脱氢酶活性。在以6-磷酸葡萄糖酸作为唯一底物的细菌裂解物孵育过程中，观察到了丙酮酸的生成，这表明存在6-磷酸葡萄糖酸脱水酶和2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶活性。通过观察在与6-磷酸葡萄糖酸孵育的细菌裂解物的时间进程中磷酸二羟丙酮的出现，证实了这些酶和磷酸丙糖异构酶的存在。通过以2-酮-3-脱氧-6-磷酸葡萄糖酸作为唯一底物孵育裂解物时丙酮酸和磷酸二羟丙酮的生成来测定醛缩酶活性。在包括新鲜分离株的野生型在内的几种细菌菌株中观察到了脱氢酶、脱水酶和醛缩酶活性。测定了这三种活性的动力学参数。通过比较离心裂解物悬浮液获得的沉淀和上清液部分中测得的活性，研究了酶的细胞定位。从剂量反应曲线确定了导致酶活性50%抑制的化合物浓度。数据表明存在两种与NAD⁺和NADP⁺活性相关的6-磷酸葡萄糖脱氢酶。使用抑制剂检测到了幽门螺杆菌和哺乳动物KDPG醛缩酶之间的差异。幽门螺杆菌中这些酶活性的存在为该细菌中恩特纳-杜德洛夫途径的存在提供了证据。
