p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac. They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif. The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding. Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques. The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac. A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM. Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132. A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75. PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type. The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42. mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar. Calorimetric measurements revealed that binding was associated with a relatively slow heat change. It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac. In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient.

CO 3؊ fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.Helicobacter pylori is a microaerophilic, gram-negative, motile, curved or spiral bacterium, isolated from the mucous layer overlying the human gastric epithelium (35,53). To date, eleven Helicobacter species have been isolated from a range of mammals (15,20,32). The organism is now accepted as the etiological agent for type B gastritis, and extensive evidence suggests a link with duodenal and gastric ulcer diseases (17,21,25,47,52). Individuals with long-term infections may also be at increased risk of developing gastric carcinoma (44).Enzyme activities identified in this bacterium include catalase, cytochrome oxidase, superoxide dismutase, and phospholipase, and it is a constitutive producer of a potent urease (18,23). Pathogenicity has been associated with adhesion to the gastric epithelium (7,24,43), urease production (36), a vacuolating cytotoxin (10,11,13), and the action of phospholipases (31). However, knowledge of the basic metabolism of H. pylori is meager. This organism exhibits limited glucose utilization (39, 41), but it is reported to contain pentose phosphate (38) and Entner-Douderoff pathways (8,42). Recently, utilization of glucose as a carbon and energy source in a defined medium has been shown (48). Active fumarate catabolism, generating malate and succinate from fumarate initially, followed by the formation of lactate, acetate, formate, and alanine, has been demonstrated (40). Suspensions of intact cells have been shown to generate lactate, ethanol, alanine, acetate, and CO 2 from pyruvate (8). This organism also requires elevated levels of CO 2 for growth, and it has been suggested that potent urease activity in part feeds this requirement (22). HCO 3 Ϫ is used in de novo synthesis of pyrimidines, but the demand for CO 2 considerably exceeds that of HCO 3 Ϫ . Gaseous CO 2 cannot be replaced by the addition of bicarbonate to the medium (22).Microorganisms exhibiting dependence on elevated CO 2 levels are termed capneic or capnophilic. Perhaps the best examples of pathogenic capnophiles are Neisseria spp. The enzymes known to be involved in CO 2 assimilation by Neisseria spp. are carbonic anhydrase (34) and phosphoenolpyruvate (PEP) carboxylase (12). Pyruvate carboxylase, PEP carboxykinase, and PEP carboxytransphosphorylase have not been dete...

The metabolic routes of substrate catabolism by intact cells of H. pylori have been investigated by 13C NMR. Real time analyses of metabolic transformations under anaerobic conditions have been obtained with dense cell suspensions incubated with 13C-labelled pyruvate and glucose. In addition, time point studies have been carried out with cells incubated under aerobic conditions. Anaerobically, pyruvate was rapidly metabolized to lactate, ethanol and acetate. In addition, alanine was produced in significant quantities by cells provided with a nitrogen source and the metabolic incorporation of nitrogen from urea was demonstrated. Under aerobic conditions acetate was the major oxidation product from pyruvate; no evidence was obtained for tricarboxylic acid cycle activity. Glucose was metabolized more slowly than pyruvate. Anaerobically, two major products were observed and identified as sorbitol and gluconate by gas chromatography/mass spectrometry. Evidence was obtained for the oxidation of glucose to acetate under aerobic conditions. The fate of the 13C label with glucose substrates labelled in different positions showed that this oxidation takes place via the Entner-Doudorof f pathway.

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953–3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por,oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. BothporD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB andoorA appeared to be lethal.