Phosphoglycerate Mutase catalyzes: 3-phosphoglycerateßà 2-phosphoglycerate

Phosphate is shifted from the hydroxyl on C3 of 3-phosphoglycerate to the hydroxyl on C2.

An active site histidine side-chain participates in phosphate transfer, by donating and accepting the phosphate. The process involves a 2,3-bisphosphate intermediate.

The Phosphoglycerate Mutase reaction is illustrated in the animation at right.

of Phosphoglycerate Mutase

9. Enolase catalyzes: 2-phosphoglycerateßàphosphoenolpyruvate + H₂O

This dehydration reaction is Mg++-dependent. 2 Mg++ ions interact with oxygen atoms of the substrate carboxyl group at the active site. The Mg++ ions help to stabilize the enolate anion intermediate that forms when a lysine side-chain amino group extracts a proton from carbon #2.

10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP à pyruvate + ATP

This transfer of phosphate from PEP to ADP is spontaneous. PEP has a larger DG of phosphate hydrolysis than ATP, because removal of phosphate from PEP yields an unstable enol, that spontaneously converts to the keto form of pyruvate (p. 602). Required inorganic cations K+ and Mg++ bind to anionic residues at the active site of Pyruvate Kinase.

Summary of Glycolysis:

The pathway continues from glyceraldehyde-3-phosphate. Recall that there are two glyceraldehyde-3-phosphate per glucose metabolized.   Balance sheet for high energy bonds of ATP: 2 ATP expended 4 ATP produced (2 from each of two 3C fragments from glucose) Net production of 2 ~P bonds of ATP per glucose.

Glycolysis Pathway(omitting H⁺):

glucose + 2 NAD⁺ + 2 ADP + 2 P_ià2 pyruvate + 2 NADH + 2 ATP

In aerobic organisms, pyruvate produced in Glycolysis is oxidized to CO₂ via Krebs Cycle, and the NADH produced in Glycolysis and Krebs Cycle is reoxidized via the respiratory chain, with production of much additional ATP.

Fermentation

Anaerobic organisms lack a respiratory chain. They must reoxidize NADHproduced in Glycolysis through some other reaction, because NAD⁺ is needed for the Glyceraldehyde-3-phosphate Dehydrogenase reaction (see above). Usually NADH is reoxidized as pyruvate is converted to a more reduced compound.

The complete pathway, including Glycolysis and the re-oxidation of NADH, is called fermentation.

For example, Lactate Dehydrogenase catalyzes reduction of the keto group in pyruvate to a hydroxyl, yielding lactate, as NADH is oxidized to NAD+. Lactate, in addition to being an end-product of fermentation, serves as a mobile form of nutrient energy, and possibly as a signal molecule in mammalian organisms. Cell membranes contain carrier proteins that facilitate transport of lactate. Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense. Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs Cycle or (in liver) converted to back to glucose via gluconeogenesis. Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment glucose to lactate and release it. Lactate taken up by adjacent neurons is converted to pyruvate that is oxidized via Krebs Cycle.

Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product. NADH is converted to NAD+ in the reaction catalyzed by Alcohol Dehydrogenase. Thiamine pyrophosphate, the cofactor for Alcohol Dehydrogenase, is discussed elsewhere.

Fermentation Pathway, from glucose to lactate (omitting H⁺):

glucose + 2 ADP + 2 P_ià2 lactate + 2 ATP

Anaerobic catabolism of glucose yields only 2 “high energy” bonds of ATP.

Regulation of Glycolysis

Glycolysis Enzyme *	DGo' (kJ/mol)	DG (kJ/mol)
Hexokinase	-20.9	-27.2
Phosphoglucose Isomerase	+2.2	-1.4
Phosphofructokinase	-17.2	-25.9
Aldolase	+22.8	-5.9
Triosephosphate Isomerase	+7.9	negative
Glyceraldehyde-3-phosphate Dehydrogenase, & Phosphoglycerate Kinase	-16.7	-1.1
Phosphoglycerate Mutase	+4.7	-0.6
Enolase	-3.2	-2.4
Pyruvate Kinase	-23.0	-13.9

*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John Wiley & Sons, New York, p. 613.

Flux through the Glycolysis pathway is regulated by control of the 3 enzymes that catalyze highly spontaneous reactions: Hexokinase, Phosphofructokinase, & Pyruvate Kinase.

• Local controlof metabolism involves regulatory effects of varied concentrations of pathway substrates or intermediates, to benefit the cell.
• Global control is for the benefit of the whole organism, & often involveshormone-activated signal cascades. Liver cells have major roles in metabolism, including maintaining blood levels various of nutrients such as glucose. Thus global control especially involves liver. Some aspects of global control by hormone-activated signal cascades will be discussed in the section on gluconeogenesis.

Hexokinase, the first step in the Glycolysis pathway, is inhibited by its product glucose-6-phosphate:

• by competition at the active site, and
• by allosteric interaction at a separate site on the enzyme.

Cells trap glucose by phosphorylating it, preventing exit on glucose carriers. Product inhibition of Hexokinase ensures that cells will not continue to accumulate glucose from the blood, if [glucose-6-phosphate] within the cell is ample.

Glucokinase is a variant of Hexokinase found in liver.

• Glucokinase has a high K_M for glucose. It is thus active only at high [glucose].
• One effect of insulin, a hormone produced in response to high blood glucose, is activation in liver of transcription of the gene that encodes the Glucokinase enzyme.
• Glucokinase is notsubject to product inhibition by glucose-6-phosphate. The liver will take up and phosphorylate glucose even when liver [glucose-6-phosphate] is high.
• Liver Glucokinase is subject to inhibition by a glucokinase regulatory protein (GKRP). The ratio of Glucokinase to GKRP in liver changes in different metabolic states, providing a mechanism for modulating rates of glucose phosphorylation.

Glucokinase, with its high KM for glucose, allows the liver to store glucose as glycogen in the fed state when blood [glucose] is high. The liver enzyme Glucose-6-phosphatase catalyzes hydrolytic release of Pi from glucose-6-phosphate. Thus glucose is released from the liver to the blood as needed to maintain blood [glucose]. The enzymes Glucokinase and Glucose-6-phosphatase, both found in liver but not in most other body cells, allow the liver to control blood [glucose].

Pyruvate Kinase, the last step of the Glycolysis pathway, is controlled in liver partly by modulation of the amount of enzyme.

• High [glucose] within liver cells causes a transcription factor carbohydrate responsive element binding protein(ChREBP) to be transferred into the nucleus, where it activates transcription of the gene for Pyruvate Kinase.
• This facilitates converting some of the excess glucose to pyruvate, which is metabolized to acetyl-CoA, the main precursor for synthesis of fatty acids, for long term energy storage.

Phosphofructokinase is usually the rate-limiting step of the Glycolysis pathway. Phosphofructokinase catalyzes: fructose-6-phosphate + ATP à fructose-1,6-bisphosphate + ADP Phosphofructokinase (PFK) is allosterically inhibited by ATP. At low concentration, the substrate ATP binds only at the active site. At high concentration, ATP binds also at a lower-affinity regulatory site, promoting the tense conformation. The tense conformation of Phosphofructokinase has a lower affinity for its other substrate, fructose-6-phosphate. Sigmoidal dependence of reaction rate on [fructose-6-phosphate] is observed at high [ATP], as depicted at right.

Effect of [ATP] on Phosphofructokinase

AMP, which is present at significant levels only when there is extensive ATP hydrolysis, antagonizes the effect of high [ATP].

Inhibition of Phosphofructokinase, the rate-limiting step of Glycolysis, when [ATP] is high, prevents breakdown of glucose, in a pathway whose main role is to make ATP. It is more useful to the cell to store glucose as glycogen when ATP is plentiful (see diagram of interconnected pathways above).

Studio Exercises:

1. Explore in the Biochemistry Simulations tutorials at right concepts of enzyme kinetics and enzyme regulation relevant to this class: · In the module on Glucokinase, compare the dependence on [glucose] of catalysis by Glucokinase and Hexokinase. · In the module on Phosphofructokinase, explore effects of varied [ATP] at zero [fructose-2,6-bisphosphate]. (The activator fructose-2,6-bisphosphate will be discussed in the section on gluconeogenesis.)

Note: Hold down the Control key while clicking on the above icon.

2. Website:Explore the following materials developed by Jon Maber.

• DIY Glycolysisis an interactive tutorial focusing on the nature of reactions in the Glycolysis pathway.
• Step by Step Glycolysis includes an animation of each step of the pathway.

DIY Glycolysis Home Page

This page can be annotated here. Select annotations above for info.

Design it Yourself Glycolysis is an educational aid which I hope will give you some insight into the function of this metabolic pathway. Step by step you will 'design' glycolysis from scratch and by doing so you can compare your selections with the real pathway which evolved billions of years ago. Please note that this resource is NOT a quiz. To get the most out of the exercise it's important that you deliberately select wrong answers to the questions that are presented to you.

Introduction

The aim of this exercise is to help you understand the processes which occur in (anaerobic) glycolysis. Starting with glucose you will choose the reaction types in sequence which will achieve the desired end point. I.e.:

1. Glucose converted to lactate.

2. ATP generated.

3. Oxidation balanced with reduction.

It is assumed that you have been introduced to glycolysis already - this application is intended to help you consolidate your understanding. You can still work through the exercise even if you are new to glycolysis by using a lot of trial and error.

The Task Ahead

In the transformation of glucose to lactate the hexose is split into two halves and the atoms are rearranged. The empirical formulae of glucose and lactic acid are;

So it's possible to make two moles of lactic acid from one mole of glucose with no net addition or removal of atoms.

Although lactic acid has the right empirical formula for a monosacharide carbon 1 is more oxidised and carbon 3 is less oxidised than the closest monosacharide - glyceraldehyde. So you need to split glucose into two three carbon monosacharides and then rearrange.

Glyceraldehyde Lactate

Keeping Track

You need to keep track of the amount of ATP you have made and the level of oxidation/reduction. A status line at the bottom of the page will do this for you. You will 'spend' ATP if you use it to phosphorylate and 'earn' ATP if you dephosporylate the substrate by transfering the phosphate group to ADP. Redox is reckoned as the number of electrons removed from glucose.