The main method of energy transfer within the cell is apparently dependent upon Thioester and phosphate compounds. There are two types of energy bound up in ester linkage. Most ester linkages contain a small amount of energy and are relatively stable.
There are a few ester linkages, however, that is relatively unstable and contains a much higher energy level. Ordinary ester linkages contain in the neighbourhood of 3000 calories of energy per mole, but the high energy (or energy-rich) bond contains between 7000 and 14,000 calories.
Strictly speaking, it is not correct to say that the compound possesses this amount of energy but that this amount of free energy is released when the bond is broken, usually by hydrolysis.
Phosphorus appears to play a universal role as a means of energy transfer in living forms, since its function has been apparent in all forms investigated. Designation of energy-rich phosphate (~ P) is misleading, however, because some energy liberated by hydrolysis of the phosphate bond doubtless resides in other linkages.
Six carbon-linked, energy-rich compounds of the hydroxyl ester group are known. Each consists of a phosphate esterified to a carbon that also contains a double-bond linkage to another carbon, nitrogen, or oxygen.
The six compounds are creatine phosphate, arginine phosphate, phosphoenolpyruvate, phosphoglyceryl phosphate, acetyl phosphate, and the phosphate-linked groups. The principal function of the first two compounds is energy storage within the cell, and the last four compounds are primarily involved in metabolic energy production and transformation.
The phosphate-linked group consists of triphosphates of purine and pyrimidine bases with attached ribose. These are known as adenosine (ATP), guanosine (GTP), uridine (UTP), inosine (ITP), thymidine (TTP), and cytidine (CTP) triphosphates.
Energy from phosphate bounds maybe utilized in forming carbon-to-carbon linkage and thus brings about synthesis. ATP, as shown earlier, is more often found in metabolism, particularly in the glycolysis of carbohydrates.
The energy-rich phosphate is located next to another phosphate that contains a double bond. This compound contains two energy-rich bonds, shown by and one phosphate may be lost to give ADP plus inorganic phosphate. If a second energy-rich bond is lost, the compound becomes adenosine monophosphate (AMP), which is one of the nucleotides.
High energy thioester linkages are formed from both coenzyme A and lipoic acid. These will be referred to later, when pyruvate breakdown and fat metabolism are described.
Energy is utilized and enzyme actions are involved in the initiation of metabolism of simple sugars:
Glucose is utilized by many microorganisms, and during one type of metabolic process, two molecules of pyruvate are formed from each molecule of glucose. This may be accomplished by more than one method, and many organisms carry out the process.
The classical illustration of conversion of glucose to pyruvate is that accomplished fermentatively by yeast cells and sometimes termed the Embden Meyerhof scheme or pathway.
This conversion system will be outlined and some other methods related to it. The student should strive at this point to master the steps in conversion and to visualize enzyme actions and energy changes involved in each step rather than to memorize an array of formulas and names that may be meaningless unless understood.
The metabolic chart should be checked at each step and an overall picture pieced together as the student reads the following description.
Glucose (alpha-D-glucopyranose) is esterified in the number 6 position to form glucose-6-phosphate. The enzyme glucokinase, or glucose-6-phosphatase, catalyzes this action, Mg++ is necessary for activation, and energy for the step is supplied by the conversion of ATP to ADP.
Reversibility of this action has been demonstrated, but it is of questionable metabolic significance in the process described here, since the reaction moves principally in one direction.
Glucose-6-phosphate is next converted into fractose-6- phosphate by the shifting of hydrogen from carbon atoms 2 to carbon atom 1. These compounds are in equilibrium, energy involved in the shift is small, and the reaction is catalyzed by the enzyme phospho- glucoisomerase. Actually, about 70% remains as glucose-6-phosphate, and only 30% is converted unless further reactions occur.
Another phosphate is esterified onto fructose-6-phosphate at the net step to form fructose- 1, 6-diphosphate. Here, the energy for esterification is characteristically derived by the conversion of ATP to ADP, and the step is catalyzed by fructose-1, 6-diphosphate and phosphofructokinase. Mg++ is essential as an activator. Energy for this conversion may also be supplied, in certain reactions, by either 1TP or UTP.
Fructose-1, 6-dphosphate is broken into two units of three carbons each by the action of a desmolase enzyme fructoaldolase. The resulting compounds from this action are dihydroxyacetone phosphate and glyceraldehyde 3-phosphate little energy is involved in this reversible reaction.
These units are interconverted by this action of an enzyme triose isomerase although much more dihydroxyacetone phosphate is present. Although the conversion of glyceraldehyde-3-phosphate to 1, 3- diphosphoglycerate involves both oxidation and phosphorylation, reactions involved in the conversion are apparently by a single enzyme.
At the beginning of the glucose-yeast fermentation reactions, glycerol is formed, but the amount is small in uninhibited reactions. It appears that glycerol is formed by the conversion of D-glyceraldehyde- 3-phosphate through the intermediate glycerol phosphate.
Substrate phosphorylation energy is produced in the conversion of phosphoglycerate to pyruvate:
In the presence of proper ingredients, including inorganic phosphate, the reaction of glyceraldhyde phosphate proceeds mainly in another direction. A molecule of inorganic phosphate is esterified at the number 1 position to form 1, 3-diphosphoglycerate.
As this reaction proceeds, more dihydroxyacetone phosphate are converted into 1, 3-diphosphoglycerate, and the process continues. As inorganic phosphate is esterified onto the phosphoglycerate, two hydrogens are given off, and this is a reaction of tremendous importance.
These hydrogens may be utilized in the reduction of organic compounds in fermentation and result in small energy transformations, or they may combine with oxygen in respiration to produce considerable energy, as will be shown in the following chapter.
Energy of the energy-rich bond next to the double- bonded oxygen in 1-3; diphosphorglycerate was derived from dehydrogenation of phosphoglyceraldehyde.
The hydrogen air is picked up by the coenzyme nicotinamide adenine dinucleotide (NAD), which functions in conjunction with the catalyzing phosphoglyceraldehyde dehydrogenase enzyme.
For hydrogen transfer in this and many other reactions, NAD or NADP is essential as accompanying coenzymes. The coenzyme NADP contains a third phosphate esterified onto the carbon 2 of the ribose adjoining the adenine.
When the preceding reaction is reversed, phosphate is given off and NADH plus H+ gives up its hydrogen and is oxidized to NAD+. The importance of this reaction will become apparent when different metabolic pathways of this system are studied.
Phosphate is next removed from the number 1 position of diphosphorglycerate, and this action is accompanied by the formation of ATP from ADP, which entails the production of an energy-rich phosphate bond by substrate phosphorylation.
The enzyme may be recognized as 3-phosphoglycerate-l-kinase. The product of this reaction is 3-phospho- glycerate, and the phosphate mutates, by action of 2, 3-phosphoglycerate mutase, to position 2, forming 2-phosphoglycerate.
The steps by which 3-phosphoglycerate is transformed into 2-phosphoglycerate apparently involve the formation of the 2, 3-diphosphorylated derivative, with both phosphate esters containing low calorie bonding.
The diphospho- compound then given up its phosphate in the number 3 position to phosphorylate, another 3-phospho- compound in conjunction with the enzyme phosphoglycerate mutase.
Water is removed from this compound by the action of enolase to form phospho-enol-pyruvate with a high energy phosphate, because it is joined next to a carbon with a double bond. Magnesium is essential as an enzyme activator for the last three steps.
Phospho-enol-pyruvate is converted to enol-pyruvate by the last of phosphate, since the phosphate linkage contains high energy, and ADP is converted into ATP in the process.
The major portion of enol-pyruvate thus formed breaks into acetaldehyd and carbon dioxide. The enzyme that catalyzes this reaction is decarboxi use. Thiamine pyrophosphate, a coenzyme, is essential for functioning of decarboxylase.
This coenzyme is built around the vitamin thiamine and is sometimes called cocarboxylase. Some of the enol-pyruvate, however, may combine with carbon dioxide to form oxalacetate which may in turn be converted into malate, succinate, or aspartate.
The reduced coenzyme NADH plus H+ may now reduce dihydroxyacetone phosphate to produce phosphoglycerol, which in turn is converted into glycerol. Another action is the reduction of the acetaldehyde formed in the foregoing reaction to ethyl alcohol, is shown. When fermentation begins, glycerol is formed, but as acetaldehyde becomes available, it is more readily reduced and the main product formed is ethyl alcohol.
If yeast juice is placed in the sugar solution and inorganic phosphate (Pi) is furnished, the phosphate is esterified to the hexose and hexose-1, and 6-diphosphate accumulates. Yeast may produce a small amount of ethyl alcohol when suspended in distilled water. The alcohol thus formed results from the breakdown of glycogen stored in the yeast cells.
The logical conclusion after discovery of the Embden-Meyerhof system of fermentation was that the process was the same in all microorganisms. Many microorganisms utilize this pathway, but others do not possess the key enzyme, aldolase, for the splitting of 1, 6-fructose diphosphate.
Pentoses, as well as hexoses, may also be metabolized, and a different conversion scheme is necessary. One main alternate conversion pathway is shown in the Warburg-Dickens-Horecker system (also called the monophosphate shunt, or the pentose phosphate system).
There are many other carbohydrate metabolic pathways. Two classical systems may combine to produce results not shown by either, but vast arrays of enzymes in microorganisms and their development in nature would suggest variations, although some pathways undoubtedly represent major metabolic scheme.
One example of a different type of fermentation is furnished by Pseudomonas lindneri, which ferments glucose or fructose to 45% ethanol, 45% carbon dioxide, and 7% lactate.
The C1 in the hexose, in this type of fermentation is converted into CO2, which is in contrast to the yeast type of fermentation in which C3 and C4 are converted to CO2 In the overall conversion it may be noted that C3 and C4 are converted to CO2, in contrast to the conversion of C3 and C4 by yeast. Likewise alcohol is formed from the C2 — C3 skeleton instead of the C1 — C2, as in yeast fermentation, but the C5 — C6 skeleton is converted to alcohol in either case.
In the just-described conversion, glucose-6-phosphate is changed to gluconate-6- phosphate, which is dehydrating to form 2-keto, 3- deoxygluconate-6-phosphate. The next reaction splits this compound to glyceraldehyde phosphate and pyruvate. The glyceraldehyde phosphate is metabolized to pyruvate, and pyruvate is converted into ethanol and carbon dioxide.
One molecule of pyruvate arises, as in the yeast fermentation, from glyceraldehyde phosphate, but the other comes directly from the hexonic acid, and substrate phosphorylation does not occur in this conversion.
In yeast fermentation, as shown earlier, two net high energy bonds are gained by substrate phosphorylation for each hexose fermented.
In conversions carried out by P. lindneri, however, only the number 6 position of the hexose is phosphorylated, and only two total energy bonds are gained. The net gain is only one high energy bond.
Bacterial alcohol fermentations in general follow hexose monophosphate instead of diphosphate pathways and convert only one half as many ADP molecules to ATP as does yeast when both ferment equal moles of hexose.
Two other examples of bacterial alcohol fermentation serve to illustrate divergent pathways. Leuconostoc produces lactate and alcohol from hexoses by way of gluconate-6-phosphate.
In this case, carbon 1 is split off to form carbon dioxide, carbons 2 and 3 form the skeleton for ethanol, and carbons 4, 5, and 6 form the skeleton for lactate.
These reactions with the P. lindneri type of fermentation. When pentoses are fermented by Enterobacteriaceae, end products are the same as when hexoses are fermented. Xylulose-5-phosphate is split into glyceraldehyde phosphate and glycolic aldehyde.
Glycolic aldehyde is transferred to ribsose-5-phosphate by the action of transketolase to form sedoheptulose-7- phosphate. Sedoheptulose-7-phosphate is broken into dihydroxyacetone and erythrose 4-phosphate.
Erythrose 4-phosphate combines with another glycolic aldehyde molecule to form fructose phosphate, which, after phosphorylation, is broken into dihydroxyacetone phosphate and glyceraldehyde phosphate. Three molecules of ribose become converted into molecules of glyceraldehyde phosphate, one molecule of dihydroxyacetone, and one molecule of dihydroxyacetone phosphate.