Key knowledge base & conceptual questions
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1 Key knowledge base & conceptual questions Why is it said the pentose phosphate pathway is the major source of reducing power? What are the differences, in structure and in function, between NADH and NADPH? Outline the pentose phosphate path, using glucose 6-phosphate, fructose 6-phosphate, glyceraldehyde 3-phosphate, ribose 5- phosphate, pyruvate, phosphoenolpyruvate, and oxaloacetate (not all compounds are needed to answer the question). Be able to recognize the reactions catalyzed by transaldolases and transketolases. Know how they are involved in interconverting intermediates in the pentose phosphate pathway and glycolysis. Why is the reversibility of transaldolase-catalyzed and transketolasecatalyzed reactions important in the linkage of the pentose phosphate shunt, glycolysis, and gluconeogenesis? In which compartment of eukaryotic cells do gluconeogenesis and the pentose phosphate pathways occur? Understand why all of these reactions do not occur in the same compartment. Be able to explain G6PD genotype, phenotype, and clinical observations.
2 Gluconeogenesis anabolic process by which glucose is synthesized PPP from smaller molecules such as lactate and pyruvate glucose important for maintenance of blood glucose levels within critical limits Glycolysis Glucose NADH + H + + ATP Pyruvate pyruvate Gluconeogenesis Overview: Introduction
3 PPP: Overview Primary functions of pathway: provide ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids generate NADPH for reductive biosynthesis reactions within cells
4 PPP: Irreversible Oxidative rxns Product = 6-phosphogluconolactone glucose 6-phosphate 6-phosphogluconate ribulose 5-phosphate NADP + coenzyme NADPH? If cellular NADPH NADP + G6PD activity ratio low? glucose 6-phosphate dehydrogenase (G6PD) 6-phosphogluconolactone dehydrogenase 6-phosphogluconolactone hydrolyase
5 PPP: Irreversible Oxidative rxns glucose 6-phosphate 6-phosphogluconate ribulose 5-phosphate 6-phosphogluconolactone glucose 6-phosphate dehydrogenase (G6PD) 6-phosphogluconolactone hydrolyase 6-phosphogluconolactone dehydrogenase
6 (reversible) PPP: Non-oxidative rxns
7 PPP: Non-oxidative rxns (reversible) transketolase (TPP) 2-C transfer rxn transaldolase 3-C transfer rxn
8 PPP: Irreversible Oxidative rxns glucose 6-phosphate 6-phosphogluconate ribulose 5-phosphate reductive anabolic pathways glucose 6-phosphate dehydrogenase (G6PD) 6-phosphogluconolactone dehydrogenase
9 H 2 N O N O H 2 C NADPH O N N OH N N OH O PO 2 PO 2 O CH 2 NH 2 OPO 3 OH O NADH H 2 N N O H 2 C O N N OH N N OH O PO 2 PO 2 O CH 2 NH 2 OH OH
10 NADPH: oxidative stress (reduced) v glutathione reductase (oxidized) NADP + NADPH + H + NADPH indirectly provides electrons for hydrogen peroxide reduction. glutathione
11 NADPH oxidase: microbial killing NADPH oxidase is required in oxygen-dependent phagocytosis Pathogen attachment & ingestion Microbial destruction
12 NADPH: nitric oxide synthesis NO - free radical reactive with O 2 & superoxide NO synthase Has four cofactors 3 types of synthases identified - Type I (neurons and epithelial cells) - Type II (macrophages, neutrophils, & hepatocytes) - Type III (endothelial cells)
13 Arg NO permeable to cell membranes NO activates guanylate cyclase, which converts GTP into cgmp NO Increased cgmp upregulates protein kinases Decreased intracellular [K + ] and [Ca 2+ ] leads to cellular hyperpolarization
14 Consequences of smooth muscle relaxation: vasodilation bronchodilation postprandial stomach relaxation Pharmaceutical targets for nitric oxide NO synthesis inhibitors NO antagonists NO mimetics Increase NO synthesis by administration of a precursor NO chemical donors (liberate NO molecules) Inhibit cgmp activation
15 PPP: Irreversible Oxidative rxns glucose 6-phosphate 6-phosphogluconate ribulose 5-phosphate G6PD monomer consists of ~500 residues (59 kda) Active human G6PD in dimertetramer equilibrium glucose 6-phosphate dehydrogenase (G6PD) Physiological significance of interconversion unclear
16 Inborn errors in CHO metabolism G6PD deficiency - prevalent enzyme abnormality in humans (1956) Populations in tropics/subtropics of Africa and Asia, Mediterranean, and Middle East X-linked inherited condition
17 G6PD deficiency family of 400 G6PD gene point mutations
18 By what means can G6PD point mutations disrupt function?
19 G6PD deficiency Genomic and structural information at the biochemical level critical for understanding disease 127 mutation sites 60 known class I mutations
20 By what means can G6PD point mutations disrupt function? Substrate-binding site Cofactor-binding site Protein folding/stability Oligomerization
21 G6PD deficiency Genomic and structural information at the biochemical level critical for understanding disease 127 mutation sites 60 known class I mutations
22 By what means can G6PD point mutations disrupt function? Substrate-binding site Cofactor-binding site Protein folding/stability Oligomerization In what part of the protein are the point mutations for G6PD A-? For G6PD Mediterranean?
23 Human G6PD monomer Au et al. (2000) Structure 8, 826 active site at amino end cofactor binding site at carboxy end
24 Human G6PD dimer Au et al. (2000) Structure 8, 826 active site at amino end cofactor binding site at carboxy end dimerization interface at carboxy end
25 Human G6PD dimer Au et al. (2000) Structure 8, 826 Class I mutations are altered residues Mutations at N-terminus are not deleterious
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