Lecture 14 Summary

The oxidative reactions of the pentose phosphate pathway. Glucose 6-phosphate dehydrogenase [EC 1.1.1.49] - specificity for NADP⁺. Mechanism of 6-phosphogluconate dehydrogenase [EC 1.1.1.44] - analogy to isocitrate dehydrogenase. Role of TPP in transketolase mechanism.

Enzymes participating in the pentose phosphate pathway

Enzyme [abbreviation, EC number] - pathway (other than PPP)

• Glucose 6-phosphate dehydrogenase [G6PDH, EC 1.1.1.49] - (specific to PPP, oxidative phase)
• Gluconolactonase [EC 3.1.1.17] - (specific to PPP, oxidative phase)
• Phosphogluconate dehydrogenase (decarboxylating) [EC 1.1.1.44] - (specific to PPP, oxidative phase)
• Transaldolase [TA, EC 2.2.1.2] - Calvin cycle (in plants); aldolase [EC 4.1.2.13] - gluconeogenesis/glycolysis
• Transketolase [TK, EC 2.2.1.1] - Calvin cycle (in plants)
• Phosphopentose isomerase [EC 5.3.1.6] - Calvin cycle (in plants)
• Phosphoribulose epimerase [EC 5.1.3.1] - Calvin cycle (in plants)
• Phosphoglycerate kinase [PGK, EC 2.7.2.3] - gluconeogenesis/glycolysis
• Glyceraldehyde 3-phosphate dehydrogenase [GAPDH, EC 1.2.1.12] - gluconeogenesis/glycolysis
• Triose phosphate isomerase [TIM, EC 5.3.1.1] - gluconeogenesis/glycolysis
• Fructose 1,6-bisphosphatase [FBPase, EC 3.1.3.11] - gluconeogenesis
• PFK: phosphofructokinase [EC 2.7.1.11] - glycolysis
• PGI: phosphoglucose isomerase [EC 5.3.1.9] - gluconeogenesis/glycolysis

Transketolase and transaldolase: Mechanism and clinical aspects

Transketolase [EC 2.2.1.1] uses TPP as a catalytic cofactor in its mechanism, and TPP forms various covalent intermediates with TK substrates. The TPP in transketolase is able to acquire, from a ketose, a glycoaldehyde unit that is then passed along to an aldose. The ylide form of the cofactor makes a nucleophilic attack on the carbonyl carbon of the substrate ketose. The bond to the adjacent carbon atom is then cleaved from this intermediate, similar to decarboxylation of an a-keto acid, leaving the two-carbon glycoaldehyde fragment. This second covalent intermediate behaves as a nucleophile toward the substrate aldose. Resonance-stabilized intermediates that react like carbanions toward electrophilic carbonyl carbons are formed in both the transketolase and the transaldolase [EC 2.2.1.2] mechanisms.

Defective transaldolase is linked in humans to cirrhosis of the liver. Genetic lesions that result in much lower affinity of transketolase for its cofactor TPP potentiates the symptoms of Wernicke-Korsakoff syndrome. These include severe memory loss, mental confusion, and even partial paralysis. This malady also afflicts alcoholics who suffer from thiamine deficiency. (More information from the National Institute of Neurological Disorders and Stroke.)

Role of oxidative branch of the pentose phosphate pathway

NADPH is of great importance in meeting cellular needs for reductive biosyntheses and coping with oxidative stress. Bear in mind that despite their close structural similarity, the difference between them is recognized in vivo, and NADH and NADPH constitute separate pools of pyridine dinucleotide electron carriers. Furthermore, in eukaryotic cells, compartmentalization allows cells to maintain transmembrane differences in redox potential. For example the [NADPH] / [NADP⁺] ratio may be maintained at a rather high level in the cytosol, promoting biosynthesis, while the cytosolic [NADH] / [NAD⁺] ratio is quite low. Within the mitochondrial matrix, the latter ratio can differ still. One of the primary sources of NADPH is the oxidative branch of the pentose phosphate pathway.

Two reactions, those catalyzed by glucose 6-phosphate dehydrogenase [G6PDH, EC 1.1.1.49] and phosphogluconate dehydrogenase (decarboxylating) [EC 1.1.1.44] involve NADPH production. Not only is glucose 6-phosphate dehydrogenase capable of discriminating between NADP⁺ and NAD⁺, its activity is quite sensitive to NADP⁺ levels. The product of the latter reaction, ribulose 5-phosphate, is readily converted to ribose 5-phosphate, a nucleotide precursor. Therefore cells with a high demand for nucleic acid synthesis (e.g. those undergoing rapid replication) may meet it in large measure via this sequence of several reactions.

Study questions

• Work out the path that regenerates glucose 6-phosphate from ribose 5-phosphate.
• Explain the role of TPP in the transketolase mechanism.
• Distiguish between NADH and NADPH: Structure, biochemical role, biological context.
• Explain the biological circumstances that place a demand on flux through the oxidative part of the PPP.
• Explain the basis of and rationale for the specificity of G6PDH for NADP⁺ vs. NAD⁺.

Page updated 12-27-06

References

1. Berg, Tymoczko, and Stryer. Biochemistry (BTS): 6th edition (2007, Freeman) Ch.20, pp.577-583.