Evidence for EnzymeTransition State Complementarity
The transition state of a reaction is difficult to study because it is so short-lived. To understand enzymatic catalysis, however, we must dissect the interaction between the enzyme and this ephemeral moment in the course of a reaction. Complementarity between an enzyme and the transition state is virtually a requirement for catalysis, because the energy hill upon which the transition state sits is what the enzyme must lower if catalysis is to occur. How can we obtain evidence for...
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Trace the pathway of each precursor through gluconeogenesis. Indicate the location of 14C in all intermediates and in the product, glucose. 17. Pathway of CO2 in Gluconeogenesis In the first bypass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate, pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently decar-boxylated by PEP carboxykinase to yield phosphoenolpyru-vate. The observation that the addition of CO2 is directly followed by the...
Info Kih
FIGURE 10-6 Some common types of storage and membrane lipids. All the lipid types shown here have either glycerol or sphingosine as the backbone pink screen , to which are attached one or more long-chain alkyl groups yellow and a polar head group blue . In triacyl-glycerols, glycerophospholipids, galactolipids, and sulfolipids, the alkyl groups are fatty acids in ester linkage. Sphingolipids contain a single fatty acid, in amide linkage to the sphingosine backbone. The membrane lipids of...
Color Blindness John Daltons Experiment from the Grave
The chemist John Dalton of atomic theory fame was color-blind. He thought it probable that the vitreous humor of his eyes the fluid that fills the eyeball behind the lens was tinted blue, unlike the colorless fluid of normal eyes. He proposed that after his death, his eyes should be dissected and the color of the vitreous humor determined. His wish was honored. The day after Dalton's death in July 1844, Joseph Ransome dissected his eyes and found the vitreous humor to be perfectly colorless....
Some Homopolysaccharides Are Stored Forms of Fuel
The most important storage polysaccharides are starch in plant cells and glycogen in animal cells. Both poly-saccharides occur intracellularly as large clusters or granules Fig. 7-14 . Starch and glycogen molecules are heavily hydrated, because they have many exposed hydroxyl groups available to hydrogen-bond with water. Most plant cells have the ability to form starch, but it is FIGURE 7-14 Electron micrographs of starch and glycogen granules. a Large starch granules in a single chloroplast....
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Native DNA undergoes reversible unwinding and separation of strands melting on heating or at extremes of pH. DNAs rich in GqC pairs have higher melting points than DNAs rich in A T pairs. Denatured single-stranded DNAs from two species can form a hybrid duplex, the degree of hybridization depending on the extent of sequence similarity. Hybridization is the basis for important techniques used to study and isolate specific genes and RNAs. DNA is a relatively stable polymer. Spontaneous reactions...
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FIGURE 13-1 Chemical basis for the large free-energy change associated with ATP hydrolysis. The charge separation that results from hydrolysis relieves electrostatic repulsion among the four negative charges on ATP. The product inorganic phosphate P is stabilized by formation of a resonance hybrid, in which each of the four phosphorus-oxygen bonds has the same degree of double-bond character and the hydrogen ion is not permanently associated with any one of the oxygens. Some degree of resonance...
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FIGURE 13-11 Nucleoside triphosphates in RNA synthesis. With each nucleoside monophosphate added to the growing chain, one PP, is released and hydrolyzed to two Ph The hydrolysis of two phospho-anhydride bonds for each nucleotide added provides the energy for forming the bonds in the RNA polymer and for assembling a specific sequence of nucleotides. molecules results in the sliding of myosin fibrils along actin filaments see Fig. 5-32 , which translates into macroscopic contraction of the...
Metabolic Control Analysis Suggests a General Method for Increasing Flux
How could an investigator engineer a cell to increase the flux through one pathway without altering the concen- trations of other metabolites or the fluxes through other pathways More than two decades ago Henrik Kacser predicted, on the basis of metabolic control analysis, that this could be accomplished by increasing the concentrations of every enzyme in a pathway. The prediction has been confirmed in several experimental tests, and it also fits with the way cells normally control fluxes...
Light Hyperpolarizes Rod and Cone Cells of the Vertebrate Eye
In the vertebrate eye, light entering through the pupil is focused on a highly organized collection of lightsensitive neurons Fig. 12-31 . The light-sensing cells are of two types rods about 109 per retina , which sense low levels of light but cannot discriminate colors, and cones about 3 X 106 per retina , which are less sensitive to light but can discriminate colors. Both cell types are long, narrow, specialized sensory neurons with two distinct cellular compartments the outer segment...
Pyruvate Is the Terminal Electron Acceptor in Lactic Acid Fermentation
When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD is regenerated from NADH by the reduction of pyruvate to lactate. As mentioned earlier, some tissues and cell types such as erythrocytes, which have no mitochondria and thus cannot oxidize pyruvate to CO2 produce lactate from glucose even under aerobic conditions. The reduction of pyruvate is catalyzed by lactate dehydrogenase, which forms the l isomer...
Xylulose 5Phosphate Is a Key Regulator of Carbohydrate and Fat Metabolism
Another recently discovered regulatory mechanism also acts by controlling the level of fructose 2,6-bisphos-phate. In the mammalian liver, xylulose 5-phosphate see Fig. 14-23 , a product of the hexose monophosphate pathway, mediates the increase in glycolysis that follows ingestion of a high-carbohydrate meal. The xylulose 5-phosphate concentration rises as glucose entering the liver is converted to glucose 6-phosphate and enters both the glycolytic and hexose monophosphate pathways. Xylulose...
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Source The Huntington's Disease Collaborative Research Group. 1993 A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971-983. 6. Using PCR to Detect Circular DNA Molecules In a species of ciliated protist, a segment of genomic DNA is sometimes deleted. The deletion is a genetically programmed reaction associated with cellular mating. A researcher proposes that the DNA is deleted in a type of recombination called...
SUMMARY 154 Coordinated Regulation of Glycogen Synthesis and Breakdown
Glycogen phosphorylase is activated in response to glucagon or epinephrine, which raise cAMP and activate PKA. PKA phosphorylates and activates phosphorylase kinase, which converts glycogen phosphorylase b to its active a form. Phosphoprotein phosphatase 1 PP1 reverses the phosphorylation of glycogen phosphorylase a, inactivating it. Glucose binds to the liver isozyme of glycogen phosphorylase a, favoring its dephosphorylation and inactivation. Glycogen synthase a is inactivated by...
Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates
As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions Fig. 16-15 Table 16-2 . Under normal circumstances, the reactions by which cycle intermediates are siphoned off into other pathways and those by which they are replenished are in dynamic balance, so that the concentrations of the citric acid cycle intermediates remain almost constant. TABLE 16-1 Stoichiometry of Coenzyme Reduction and ATP Formation in the...
Pyruvate Kinase Is Allosterically Inhibited by ATP
At least three isozymes of pyruvate kinase are found in vertebrates, differing in their tissue distribution and their response to modulators. High concentrations of ATP, acetyl-CoA, and long-chain fatty acids signs of abundant energy supply allosterically inhibit all isozymes of pyruvate kinase Fig. 15-19 . The liver isozyme L form , but not the muscle isozyme M form , is subject to further regulation by phosphorylation. When low blood glucose causes glucagon release, cAMP-dependent protein...
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FIGURE 10-18 Arachidonic acid and some eicosanoid derivatives. a In response to hormonal signals, phospholipase A2 cleaves arachidonic acid-containing membrane phospholipids to release arachidonic acid arachidonate at pH 7 , the precursor to various eicosanoids. b These compounds include prostaglandins such as PGE-i, in which C-8 and C-12 of arachidonate are joined to form the characteristic five-membered ring. In thromboxane A2, the C-8 and C-12 are joined and an oxygen atom is added to form...
Lipids
10.2 Structural Lipids in Membranes 348 10.3 Lipids as Signals, Cofactors, and Pigments 357 The fatty substance, separated from the salifiable bases, was dissolved in boiling alcohol. On cooling, it was obtained crystallized and very pure, and in this state it was examined. As it has not been hitherto described I purpose to call it margarine, from the Greek word signifying pearl, because one of its characters is to have the appearance of mother of pearl, which it communicates to several of the...
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FIGURE 17-4 Entry of glycerol into the glycolytic pathway. in the outer mitochondrial membrane, the acyl-CoA synthetases, which promote the general reaction Fatty acid CoA ATP yz fatty acyl-CoA AMP PPi Thus, acyl-CoA synthetases catalyze the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl-CoA, coupled to the cleavage of ATP to AMP and PPj. Recall the description of this reaction in Chapter 13, to illustrate how the...
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9. Base Pairing in DNA In samples of DNA isolated from two unidentified species of bacteria, X and Y, adenine makes up 32 and 17 , respectively, of the total bases. What relative proportions of adenine, guanine, thymine, and cytosine would you expect to find in the two DNA samples What assumptions have you made One of these species was isolated from a hot spring 64 C . Suggest which species is the ther-mophilic bacterium. What is the basis for your answer 10. Solubility of the Components of DNA...
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9. Cloning in Plants The strategy outlined in Figure 9-28 employs Agrobacterium cells that contain two separate plasmids. Suggest why the sequences on the two plasmids are not combined on one plasmid. 10. DNA Fingerprinting and RFLP Analysis DNA is extracted from the blood cells of two different humans, individuals 1 and 2. In separate experiments, the DNA from each individual is cleaved by restriction endonucleases A, B, and C, and the fragments separated by electrophoresis. A hypothetical map...
Myoglobin Has a Single Binding Site for Oxygen
Myoglobin Mr 16,700 abbreviated Mb is a relatively simple oxygen-binding protein found in almost all mammals, primarily in muscle tissue. As a transport protein, it facilitates oxygen diffusion in muscle. Myoglobin is particularly abundant in the muscles of diving mammals such as seals and whales, where it also has an oxygen-storage function for prolonged excursions undersea. Proteins very similar to myoglobin are widely distributed, occurring even in some single-celled organisms. Myoglobin is...
Info Kao
constant with units of s . There is an upper limit to kcat Km, imposed by the rate at which E and S can diffuse together in an aqueous solution. This diffusion- s_1, and many enzymes near this range Table 6-8 . Such en- V0 in this case depends on the concentration of two re-actants, Et and S therefore this is a second-order rate equation and the constant kcat Km is a second-order rate controlled limit is 108 to 109 m have a kcat Km zymes are said to have achieved catalytic perfection. Note that...
Myoglobin Provided Early Clues about the Complexity of Globular Protein
Q Protein Architecture Tertiary Structure of Small Globular Proteins, II. Myoglobin The first breakthrough in understanding the three-dimensional structure of a globular protein came from x-ray diffraction studies of myoglobin carried out by John Kendrew and his colleagues in the 1950s. Myoglobin is a relatively small Mr 16,700 , oxygen-binding protein of muscle cells. It functions both to store oxygen and to facilitate oxygen diffusion in rapidly contracting muscle tissue. Myoglobin contains a...
Box 153 Working In Biochemistry
Metabolic Control Analysis Quantitative Aspects The factors that influence the flow of intermediates flux through a pathway may be determined quantitatively by experiment and expressed in terms useful for predicting the change in flux when some factor involved in the pathway changes. Consider the simple reaction sequence in Figure 1, in which a substrate X say, glucose is converted in several steps to a product Z perhaps pyruvate, formed glycolytically . A later enzyme in the pathway is a...
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tFBPase-2 4Pyruvate 4-PFK-2 kinase L Between meals, or during an extended fast, the drop in blood glucose triggers the release of glucagon, which, acting through the cascade shown in Figure 15-25, activates PKA. PKA mediates all the effects of glucagon Fig. 15-31, bottom . It phosphorylates phosphorylase kinase, activating it and leading to the activation of glyco-gen phosphorylase. It phosphorylates glycogen synthase, inactivating it and blocking glycogen synthesis. It phos-phorylates PFK-2...
The Elasticity Coefficient Is Related to an Enzymes Responsiveness to Changes
A second parameter, the elasticity coefficient, e, expresses quantitatively the responsiveness of a single enzyme to changes in the concentration of a metabolite or regulator it is a function of the enzyme's intrinsic kinetic properties. For example, an enzyme with typical Michaelis-Menten kinetics shows a hyperbolic response to increasing substrate concentration Fig. 15-35 . At low concentrations of substrate say, 0.1 Zm each increment in substrate concentration results in a comparable...
Amplification of the Visual Signal Occurs in the Rod and Cone Cells
Several steps in the visual-transduction process result in great amplification of the signal. Each excited rhodopsin molecule activates at least 500 molecules of transducin, each of which can activate a molecule of PDE. This phos-phodiesterase has a remarkably high turnover number, each activated molecule hydrolyzing 4,200 molecules of cGMP per second. The binding of cGMP to cGMP-gated ion channels is cooperative at least three cGMP molecules must be bound to open one channel , and a relatively...
An Introduction to Enzymes
Much of the history of biochemistry is the history of enzyme research. Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach, and research continued in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts. In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by ferments. He postulated that these ferments were...
Binding Energy Contributes to Reaction Specificity and Catalysis
Can we demonstrate quantitatively that binding energy accounts for the huge rate accelerations brought about by enzymes Yes. As a point of reference, Equation 6-6 allows us to calculate that AG must be lowered by about 5.7 kJ mol to accelerate a first-order reaction by a factor of ten, under conditions commonly found in cells. The energy available from formation of a single weak interaction is generally estimated to be 4 to 30 kJ mol. The overall energy available from a number of such...
Hexokinase Isozymes of Muscle and Liver Are Affected Differently by Their
Hexokinase, which catalyzes the entry of free glucose into the glycolytic pathway, is a regulatory enzyme. There are four isozymes designated I to IV , encoded by four different genes. Isozymes are different proteins that catalyze the same reaction Box 15-2 . The predominant hexokinase isozyme of myocytes hexokinase II has a high affinity for glucose it is half-saturated at about 0.1 mm. Because glucose entering myocytes from the blood where the glucose concentration is 4 to 5 mm produces an...
Fructose 26Bisphosphate Is a Potent Regulator of Glycolysis and Gluconeogenesis
The special role of liver in maintaining a constant blood glucose level requires additional regulatory mechanisms to coordinate glucose production and consumption. When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose and to stop consuming it for its own needs. One source of glucose is glycogen stored in the liver another source is gluconeogenesis. The hormonal regulation of glycolysis and gluco-neogenesis is mediated by fructose...
Info Wtg
100-amino-acid protein would be synthesized with exquisite fidelity in about 5 seconds in a bacterial cell. A variety of new methods for the efficient ligation joining together of peptides has made possible the assembly of synthetic peptides into larger proteins. With these methods, novel forms of proteins can be created with precisely positioned chemical groups, including those that might not normally be found in a cellular protein. These novel forms provide new ways to test theories of enzyme...
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Each P-type ATPase transporter is an integral protein with ten predicted membrane-spanning regions in a single polypeptide some also have a second subunit. The P-type transporters are very widely distributed. In animal tissues, the Na K ATPase an antiporter for Na and K and the Ca2 ATPase a uniporter for Ca2 are ubiquitous P-type ATPases that maintain differences in the ionic composition of the cytosol and the extracellular medium. Parietal cells in the lining of the mammalian stomach have a...
Globular Proteins Have a Variety of Tertiary Structures
With elucidation of the tertiary structures of hundreds of other globular proteins by x-ray analysis, it became clear that myoglobin illustrates only one of many ways in which a polypeptide chain can be folded. In Figure 4-18 the structures of cytochrome c, lysozyme, and ribonuclease are compared. These proteins have different amino acid sequences and different tertiary structures, reflecting differences in function. All are relatively small and easy to work with, facilitating structural...
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1,3-Bisphosphoglycerate aG' 6.3 kJ mol This is the first of the two energy-conserving reactions of glycolysis that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid. This type of anhydride, called an acyl phosphate, has a very high standard free energy of hydrolysis AG' 49.3 kJ mol see Fig. 13-4, Table 13-6 . Much of the free energy of oxidation of the...
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cAMP-gated cation channels open. Ca2 enters, raising internal Ca2 . Ga-GTP activates adenylyl cyclase, which catalyzes cAMP synthesis, raising cAMP . cAMP-gated cation channels open. Ca2 enters, raising internal Ca2 .
Figure 3-25 Steps In Sequencing A Polypeptide
FIGURE 3-25 Steps in sequencing a polypeptide. a Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger's method for identifying the amino-terminal residue is shown here. b The Edman degradation procedure reveals the entire sequence of a peptide. For shorter peptides, this method alone readily yields the entire sequence, and step a is often omitted. Step a is useful in the case of larger polypeptides, which are often fragmented into smaller...
Coordinated Regulation of Glycogen Synthesis and Breakdown
As we have seen, the mobilization of stored glycogen is brought about by glycogen phosphorylase, which degrades glycogen to glucose 1-phosphate Fig. 15-3 . Glycogen phosphorylase provides an especially instructive case of enzyme regulation. It was one of the first known examples of an allosterically regulated enzyme and the first enzyme shown to be controlled by reversible phosphorylation. It was also one of the first allosteric enzymes for which the detailed three-dimensional structures of the...
Box 41 Working In Biochemistry
Knowing the Right Hand from the Left There is a simple method for determining whether a helical structure is right-handed or left-handed. Make fists of your two hands with thumbs outstretched and pointing straight up. Looking at your right hand, think of a helix spiralling up your right thumb in the direction in which the other four fingers are curled as shown counterclockwise . The resulting helix is right-handed. Your left hand will demonstrate a left-handed helix, which rotates in the...
The Free Energy of Hydrolysis of ATP within Cells The Real Cost of Doing
The standard free energy of hydrolysis of ATP is -30.5 kJ mol. In the cell, however, the concentrations of ATP, ADP, and Pj are not only unequal but much lower than the standard 1 m concentrations see Table 13-5 . Moreover, the cellular pH may differ somewhat from the standard pH of 7.0. Thus the actual free energy of hydrolysis of ATP under intracellular conditions AGp differs from the standard free-energy change, AG' . We can easily calculate AGp. In human erythrocytes, for example, the...
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NADH and NAD are the reduced and oxidized forms, respectively, of the coenzyme NAD. Solutions of NADH, but not NAD , absorb light at 340 nm. This property is used to determine the concentration of NADH in solution by measuring spectrophotometrically the amount of light absorbed at 340 nm by the solution. Explain how these properties of NADH can be used to design a quantitative assay for lactate dehydrogenase. 7. Relation between Reaction Velocity and Substrate Concentration Michaelis-Menten...
The Kinetic Properties of Allosteric Enzymes Diverge from MichaelisMenten
Allosteric enzymes show relationships between V0 and S that differ from Michaelis-Menten kinetics. They do exhibit saturation with the substrate when S is sufficiently high, but for some allosteric enzymes, plots of V0 versus S Fig. 6-29 produce a sigmoid saturation curve, rather than the hyperbolic curve typical of non-regulatory enzymes. On the sigmoid saturation curve we can find a value of S at which V0 is half-maximal, but we cannot refer to it with the designation Km, because the enzyme...
Water Interacts Electrostatically with Charged Solutes
Water is a polar solvent. It readily dissolves most biomolecules, which are generally charged or polar compounds Table 2-2 compounds that dissolve easily in water are hydrophilic Greek, water-loving . In contrast, nonpolar solvents such as chloroform and benzene are poor solvents for polar biomolecules but easily dissolve those that are hydrophobic nonpolar molecules such as lipids and waxes. Water dissolves salts such as NaCl by hydrating and stabilizing the Na and Cl- ions, weakening the...
Glycogenin Primes the Initial Sugar Residues in Glycogen
Glycogen synthase cannot initiate a new glycogen chain de novo. It requires a primer, usually a preformed a1n4 polyglucose chain or branch having at least eight glucose residues. How is a new glycogen molecule initiated The intriguing protein glycogenin Fig. 15-10 is both the primer on which new chains are assembled and the enzyme that catalyzes their assembly. The first step in the synthesis of a new glycogen mole cule is the transfer of a glucose residue from UDP-glucose to the hydroxyl group...
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FIGURE 13-9 Ranking of biological phosphate compounds by standard free energies of hydrolysis. This shows the flow of phosphoryl groups, represented by from high-energy phosphoryl donors via ATP to acceptor molecules such as glucose and glycerol to form their low-energy phosphate derivatives. This flow of phosphoryl groups, catalyzed by enzymes called kinases, proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low-energy phosphate compounds releases Ph...
Further Reading 1
Evolution of Catalytic Function. 1987 Cold Spring Harb. Symp. Quant. Biol. 52. A collection of excellent papers on fundamentals continues to be very useful. Fersht, A. 1999 Structure and Mechanism in Protein Science A Guide to Enzyme Catalysis and Protein Folding, W. H. Freeman and Company, New York. A clearly written, concise introduction. More advanced. Friedmann, H. ed. 1981 Benchmark Papers in Biochemistry, Vol. 1 Enzymes, Hutchinson Ross Publishing Company, Stroudsburg, PA. A collection of...
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4. Identifying the Gene for a Protein with a Known Amino Acid Sequence Using Figure 27-7 to translate the genetic code, design a DNA probe that would allow you to identify the gene for a protein with the following amino-terminal amino acid sequence. The probe should be 18 to 20 nucleotides long, a size that provides adequate specificity if there is sufficient homology between the probe and the gene. 5. Designing a Diagnostic Test for a Genetic Disease Huntington's disease HD is an inherited...
SickleCell Anemia Is a Molecular Disease of Hemoglobin
The great importance of the amino acid sequence in determining the secondary, tertiary, and quaternary structures of globular proteins, and thus their biological functions, is strikingly demonstrated by the hereditary human disease sickle-cell anemia. Almost 500 genetic variants of hemoglobin are known to occur in the human population all but a few are quite rare. Most variations consist of differences in a single amino acid residue. The effects on hemoglobin structure and function are often...
Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions
The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate PEP . This reaction cannot occur by reversal of the pyruvate kinase reaction of glycolysis p. 532 , which has a large, negative standard free-energy change and is irreversible under the conditions prevailing in intact cells Table 14-2, step . Instead, the phosphoryla-tion of pyruvate is achieved by a roundabout sequence of reactions that in eukaryotes requires enzymes in both the cytosol...