Lecture 26 Summary

Our examination of the central metabolic pathways now begins with a close look at glycolysis. Our goal will be to understand the reactions and enzymes of glycolysis on several levels: mechanistic, energetic, and structure-reactivity or structure-function relationships. We'll develop an appreciation of the logic of certain steps of the pathway, and we will go on to consider the regulation of glycolysis.

We have spent much time this semester developing the themes of structure-function relationships in proteins (enzymes in particular) and bioenergetics. We have sought to understand enzymes in terms of their kinetics, the catalytic principles they embody, and their modes of regulation. This has set the stage for us to apply this knowledge toward an understanding of metabolic pathways - the individual reactions and enzymes that catalyze them, and how the pathways are regulated at key points. We will need to draw upon our background in organic chemistry as well to truly appreciate the reactions and mechanisms of metabolism. The multiple steps of the many different metabolic pathways may seem bewildering at first, but the ultimate advantage of a mechanistic approach to metabolism is its ability to serve as a unifying principle. What may appear to be countless variety of reactions and their associated mechanisms can actually be distilled down in their essential features to a short list of reaction types and the characteristic mechanistic strategies underlying them.

Overview of glycolysis

The common, central pathway of glycolysis starts with glucose, a six-carbon monosaccharide, and converts it to two molecules of pyruvate, a three-carbon α-keto acid. As a catabolic pathway, the purpose of glycolysis is to extract energy from the breakdown of larger, more complex molecules to smaller, simpler molecules. Energetically, the net result of glycolysis is the production of two molecules of ATP per glucose molecule catabolized. Pyruvate has a variety of fates that we will consider soon, but one major generalization has to do with whether metabolism is proceeding under oxidative or anaerobic conditions. In the former case, pyruvate is oxidatively decarboxylated to yield acetyl CoA, which feeds directly into the citric acid cycle. In the latter case, pyruvate must be reduced to regenerate the oxidized form of the nicotinamide redox cofactor - NAD+. This is required for glycolysis to continue under anaerobic conditions. The operation of glycolysis anaerobically is termed fermentation. Biochemistry's roots as a science are intimately connected to early studies of fermentation. With respect to energy yield, one key point about oxidative metabolism versus strictly anaerobic metabolism is that the latter yields much less energy. There is much more energy available from the complete oxidation of glucose to six molecules each of carbon dioxide and water than the net yield of two ATP avialable from fermentation.

Principles of metabolic regulation and control

Homeostasis: Cells and organisms constitute open, nonequilibrium systems operating at steady-state

Pathway flux is regulated to meet the needs of the organism

Reciprocal regulation of opposing pathways

Mechanisms of flux control

Inputs/outputs
Regulation of enzyme activity
Substrate cycles

Let us consider two cases: reactions that operate close to equilibrium, and are therefore reversible, and reactions that are far from equilibrium, which have relatively large ΔG°′ values. In the former case, relatively minor adjustments of the balance between reactants and products can readily accomplish a switch in the direction of the reaction. Enzymes catalyzing such reactions are rarely subject to regulation. In the latter case, the reaction remains far from equilibrium over a huge range of reactant and product concentrations, so that it is practically impossible to reverse a reaction with a large and negative ΔG°′ value by lowering the reactant to product concentration ratio (Q). Hence, we term these reactions irreversible, and the enzymes that catalyze them are most important in controlling flux through the pathway of which they are part of. Recall that we defined the committed step of a pathway as the first irreversible step that is unique to that pathway. Enzymes catalyzing a committed step of a pathway are typically subject to regulation, which occurs by the means we have discussed: allosteric control, reversible covalent modification (principally phosphorylation/dephosphorylation by kinases/phosphatases), and genetic control of the enzyme levels.

For an irreversible step of a metabolic pathway, substrate levels are analogous to a water reservoir, and the enzyme is analogous to a dam that controls the rate of downstream flow. Below is a diagram of the free energy profile for a cell in which metabolic flux is from glucose to pyruvate, i.e. glycolysis is predominant. This would likely be the case if demand for ATP were high (energy charge low). Control of glycolytic flux is potentially greatest for the irreversible steps (steps 1, 3, and 10), whereas the other steps that are closer to equilibrium respond sensitively to the change in levels of their substrates. The product of reaction 1, glucose 6-phosphate, can have several possible fates, so that step 3 becomes the committed step of glycolysis.

Acetyl CoA and thioesters

Coenzyme A, a carrier of acyl groups, contains an active thiol group that forms a thioester linkage with acyl groups, forming various acyl CoA species. The structure of coenzyme A shows that it is made up of an adenine nucleotide (AMP) linked to phosphopanthetheine, derived from the B vitamin pantothenate. Acetyl CoA, the thioester formed when the acyl group is acetate, is central to the oxidative metabolism of carbohydrates, as well as that of fatty acids and amino acids.

Acyl CoAs are good acylating agents, as the thioester bond is weaker than the typical oxygen ester bond. The biochemical standard free energy of thioester hydrolysis is about the same as that for ATP hydrolysis. In the citrate synthase reaction, in which acetyl CoA condenses with oxaloacetate, the thioester first acts as an alkylating agent, then its hydrolysis contributes to driving the reaction forward.

Learning objectives

Page update in progress, 11-01-09

References