A Road Map for Cellular Respiration Cytosol Mitochondrion High-energy electrons carried by NADH High-energy electrons carried mainly by NADH Glycolysis 2 Pyruvic acid Krebs Cycle Electron Transport Glucose Figure 6.7

Respiration Overview O2 and glucose to CO2 + H2O + energy($$) C6H12O6 + O2  6CO2 + 6H2O + 38 ATP Glucose is highly reduced; contains energy Oxygen receives the electrons to form energy 4 separate reactions Glycolysis, Transition Reaction, Krebs Cycle, Electron Transport, Requires Oxygen Glucose Oxygen Carbon dioxide Water Energy

Glycolysis Most completely understood biochemical pathway Plays a key role in energy metabolism by providing significant portion of energy utilized by most organisms Splits the 6-C sugar (glyco-lysis) Generates two molecules of ATP per molecule of glucose Converts two NAD+ to NADH per molecule of glucose

Lactic Acid Fermentation Ethanol Fermentation Lactic Acid Fermentation Fermentation of glucose to ethanol: Wine making & baking both exploit this process From Lehninger Principles of Biochemistry

2 Pyruvic acid Glucose Figure 6.8

Glycolysis takes place in the cytosol of cells. Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially there is energy input corresponding to cleavage of two ~P bonds of ATP.

Hexokinase The first enzyme in the glycolysis pathway Hexokinase undergoes a dramatic conformational change upon binding glucose. Two lobes of the enzyme come together to surround glucose and exclude water from the active site. The ATP binding site is formed after glucose binds to the enzyme. "induced fit"

1. Phosphorylation by Hexokinase: Glucose + ATP  glucose-6-P + ADP The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP. ATP binds to the enzyme as a complex with Mg++.

The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP. Mg++ interacts with negatively charged phosphate oxygen atoms, providing charge compensation & promoting a favorable conformation of ATP at the active site of the Hexokinase enzyme.

The reaction catalyzed by Hexokinase is highly spontaneous. A phosphoanhydride bond of ATP (~P) is cleaved. The phosphate ester formed in glucose-6-phosphate has a lower DG of hydrolysis.

Hexokinase conformational change PDB: 2YHX and 1HKG The active site pocket changes shape upon binding glucose Only then can ATP transfer its phosphoryl group to the C6 carbon, yielding Glu-6-P + ADP

Induced fit: Glucose binding to Hexokinase stabilizes a conformation in which: the C6 hydroxyl of the bound glucose is close to the terminal phosphate of ATP, promoting catalysis. water is excluded from the active site. This prevents the enzyme from catalyzing ATP hydrolysis, rather than transfer of phosphate to glucose. 

Only then can ATP transfer its phosphoryl group to the C6 carbon, yielding Glu-6-P + ADP

An inhibitor of hexokinase Xylose can cause a similar conformational change But xylose does not get phosphorylated, so ATP hydrolysis is stimulated with phorphoryl group transfer to water (water gets in!)

It is a common motif for an enzyme active site to be located at an interface between protein domains that are connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, as substrate binding promotes a particular conformation.

Hexokinase in inhibited by G6P When there are high levels of G6P, it will bind to the active site, thus it acts like a competitive inhibitor

Hexokinase is inhibited by product glucose-6-phosphate: by competition at the active site by allosteric interaction at a separate enzyme site. 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 KM: ~20mM glucose hexokinase vs. glucokinase Both catalyze early step in breakdown of sugars hexokinase KM: ~0.15mM glucose ATP ADP + Pi glucokinase KM: ~20mM glucose

Glucokinase is a variant of Hexokinase found in liver. Glucokinase has a high KM for glucose. It is active only at high [glucose]. One effect of insulin, a hormone produced when blood glucose is high, is activation in liver of transcription of the gene that encodes the Glucokinase enzyme. Glucokinase is not subject to product inhibition by glucose-6-phosphate. Liver will take up & phosphorylate glucose even when liver [glucose-6-phosphate] is high.

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

Isomerases catalyze bond rearrangement within a molecule.

2. Isomerization by Phosphoglucose Isomerase: glucose-6-P  fructose-6-P The mechanism involves acid/base catalysis, with ring opening, isomerization, and then ring closure.

3. Phosphorylation by Phosphofructokinase : fructose-6-P + ATP  fructose-1,6-bisP + ADP The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated.

Phosphofructokinase, PFK-1 Catalyzes the committing step into glycolysis

3. Phosphorylation by Phosphofructokinase : fructose-6-P + ATP  fructose-1,6-bisP + ADP The enzyme is highly regulated. fructose-1,6-bisP, the product CANNOT be catabolized by any other pathway by glycolysis Process needs a ton of energy and is irreversible

Catalytic site Allosteric site Allosteric site Catalytic site

Phosphofructokinase, PFK-1 Has five, 5, allosteric regulators AMP, ADP, ATP, citrate and fructose 2,6-bisphosphate

Phosphofructokinase is usually the rate-limiting step of the Glycolysis pathway. Phosphofructokinase 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 low-affinity regulatory site, promoting the tense conformation.

PFK-1 Regulation AMP and ADP are activators. As ATP is consumed, ADP and sometimes AMP levels build up, triggering the need for more ATP. The enzyme is highly regulated by ATP. If there is a lot of ATP in the cell, then glycolysis is not necessary.. ATP will build at an allosteric site and inhibit binding of F6-P.

PFK Regulation Citrate – Inhibitor of PFK-1 in liver; an early intermediate of the citric acid cycle. Its presence indicates that the needs of the cell are being met by other means so glycolysis can slow down. Fructose 2,6-bisphosphate – a powerful activator of PFK-1. F26BP made when plenty of F6P, thus plenty of glucose PFKs equilibrium is towards the T state so it NEEDs F26BP to take it to R!

Inhibition of the Glycolysis enzyme Phosphofructokinase 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.

4. Cleavage by Aldolase: fructose-1,6-bisphosphate  dihydroxyacetone-P + glyceraldehyde-3-P The reaction is an aldol cleavage, the reverse of an aldol condensation.

A lysine residue at the active site functions in catalysis. The keto group of fructose-1,6-bisphosphate reacts with the e-amino group of the active site lysine, to form a protonated Schiff base intermediate. Cleavage of the bond between C3 & C4 follows.

5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P  glyceraldehyde-3-P Glycolysis continues from glyceraldehyde-3-P. TIM's Keq favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P by a subsequent spontaneous reaction allows throughput.

The ketose/aldose conversion involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase. Active site Glu (base) and His (acid) residues extract and donate protons during catalysis.

http://chemistry.umeche.maine.edu/CHY431/Enzyme3.html

2-Phosphoglycolate is a transition state analog that binds tightly at the active site of Triose Phosphate Isomerase (TIM). This inhibitor of catalysis by TIM is similar in structure to the proposed enediolate intermediate. TIM is judged a "perfect enzyme." Reaction rate is limited only by the rate that substrate collides with the enzyme.

structure is an ab barrel, or TIM barrel. In an ab barrel there are 8 parallel b-strands surrounded by 8 a-helices with short loops connecting alternating b-strands & a-helices.

TIM barrels serve as scaffolds for active site residues in a diverse array of enzymes. Residues of the active site are always at the same end of the barrel, on C-terminal ends of b-strands & loops connecting these to a-helices. There is debate whether the many different enzymes with TIM barrel structures are evolutionarily related. In spite of the structural similarities there is tremendous diversity in catalytic functions of these enzymes and little sequence homology.

Exergonic oxidation of the aldehyde in glyceraldehyde- 3-phosphate, to a carboxylic acid, drives formation of an acyl phosphate, a "high energy" bond (~P). This is the only step in Glycolysis in which NAD+ is reduced to NADH.