Biochem Test 4

List the reasons why phosphorylation is such an effective control mechanism.

1. The free energy of phosphorylation is large. 2. A phosphoryl group adds two negative charges to a modified protein. 3. A phosphoryl group can form three or more hydrogen bonds. 4. Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours. 5. Phosphorylation often evokes highly amplified effects. 6. ATP is the cellular energy currency

Define O-glycosidic and N-glycosidic bonds in terms of acetal and ketal bonds.

A bond formed between the anomeric carbon atom of a carbohydrate and the oxygen atom of an alcohol is called a glycosidic bond—specifically an O-glycosidic bond. They are prominent when carbohydrates are linked together to form long polymers and when they are attached to proteins. A bond formed between the anomeric carbon atom of a carbohydrate and the nitrogen atom of an amine is an N-gycosidic bond, such as when nitrogenous bases are attached to ribose ribose units to form nucleosides.

Describe the general properties of the fatty acid chains found in phospholipids, glycolipids, and cholesterol.

A phospholipid molecule is constructed from four components: one or more fatty acids, a platform to which the fatty acids are attached, a phosphate, and an alcohol attached to the phosphate. The fatty acid components provide a hydrophobic barrier, whereas the remainder of the molecule has hydrophilic properties that enable interaction with the aqueous environment. The platform on which phospholipids are built may be glycerol, a three-carbon alcohol, or sphingosine, a more complex alcohol. Phospholipids derived from glycerol are called phosphoglycerides. This consists of a glycerol backbone to which are attached two fatty acid chains and a phosphorylated alcohol. Cholesterol is a steroid from four linked hydrocarbon rings. A hydrocarbon tail is linked to the steroid at one end, and a hydroxyl group is attached at the other end. The orientation of the molecule is parallel to the fatty acid chains and the hydroxyl group interacts with the nearby phospholipid head groups.

Describe the composition and arrangement of the subunits of ATCase and the major features of its active site as revealed by the binding of N-(phosphonacetyl)-L-aspartate (PALA) and treatment with p-hydroxymercuribenzoate. Explain the effects of subunit dissociation on the allosteric behavior of the enzyme.

ATCase can be separated into regulatory and catalytic subunits by treatment with a mercurial compound such as p-hydroxymercuribenzoate, which reacts with sulfhydryl groups. ATCase is composed of two kinds of subunits. They differ markedly in charge and size. The larger subunit is the catalytic subunit and has catalytic activity but displays the hyperbolic kinetics of Michaelis-Menten enzymes rather than sigmoidal kinetics. The isolated catalytic subunit is unresponsive to CTP. The isolated smaller subunit can bind CTP but has no catalytic activity. Hence, it is the regulatory subunit. The catalytic subunit consists of three chains, and the regulatory subunit has two chains. P-hydroxymercuribenzoate is able to dissociate the catalytic and regulatory subunits because mercury binds strongly to the cysteine residues, displacing the zinc and preventing interactions with the catalytic chain. PALA is used to locate the active sites.

Write the basic reactions catalyzed by protein kinases and protein phosphatases.

ATP is the most common donor of phosphoryl groups. The terminal phosphoryl group of ATP is transferred to a specific amino acid of the acceptor protein or enzyme. In eukaryotes, the acceptor residue is commonly one of the three containing a hydroxyl group in its side chain. Transfers to a serine or threonine are handled by one class of protein kinases and to tyrosine by another. Dedicated protein kinases phosphorylate a single protein. Multifunctional protein kinases modify many different targets. The primary determinant of specificity is the amino acid sequence surrounding the serine or threonine phosphorylation site. Protein phosphatases reverse the effects of kinases by catalyzing the removal of phosphoryl groups attached to proteins. The enzyme hydrolyzes the bond attaching the phosphoryl group.

Explain the differences in the kinetic curves of an enzyme following Michaelis-Menten kinetics and an allosterically regulated enzyme.

Allosteric enzymes are distinguished by their response to changes in substrate concentration in addition to their susceptibility to regulation by other molecules. The curve for the rate of ATCase is a sigmoidal curve. Sigmoidal curves result from cooperation between subunits: the binding of substrate to one active site in a molecule increases the likelihood that substrate will bind to other active sites.

Define carbohydrate and monosaccharide in chemical terms.

Carbohydrates are carbon-based molecules that are rich in hydroxyl groups. Simply, carbohydrates are called monosaccharides. These simple sugars serve not only as fuel molecules but also as fundamental constituents of living systems. Monosaccharides are aldehydes or ketones that have two or more hydroxyl groups.

Describe the formation of the substrate-binding site of chymotrypsin by proteolysis of chymotrypsinogen.

Chymotrypsin's inactive precursor chymotrypsinogen is synthesized in the pancreas. The striking feature of the activation process is that cleavage of a single specific peptide bond transforms the proteins from a catalytically inactive form into one that is fully active (bw AA arg 15 and ile 16). Then, 1. The newly formed amino-terminal group of isoleucine 16 turns inward and forms an ionic bond with aspartate 194 in the interior. 2. This electrostatic interaction moves methionine 192 from deeply buried to the surface and moves residues 187 and 193 further apart. This results in the formation of the substrate specificity site for aromatic and bulky nonpolar groups. One side of this site has residues 189 and 192.

Relate the absolute configuration of monosaccharide D or L stereoisomers to those of glyceraldehyde.

Dihydroxyacetone is called a ketose because it contains a keto group whereas glyceraldehyde is called an aldose because it contains an aldehyde group.

Distinguish among enantiomers, diastereoisomers, anomers, and epimers of monosaccharides.

Enantiomers: nonsuperimposable mirror images; diastereoisomers: isomers that are not mirror images (2 C's different); anomers: isomers that differ at a new asymmetric carbon atom formed on ring closure (alpha and beta); epimers: differ at one of several asymmetric carbon atoms (3 C's)

Explain the relationship between fatty acid chain length and degree of saturation and the physical property of melting point.

Fatty acids usually contain an even number of carbon atoms typically between 14 and 24. The configuration of double bonds in most unsaturated fatty acids is cis. Unsaturated fatty acids have lower melting points than saturated fatty acids of the same length do. The melting point of polyunsaturated fatty acids are even lower. The short chain length and unsaturation enhance the fluidity of fatty acids and of their derivatives.

Define feedback inhibition and describe how ATCase uses it as a regulatory mechanism.

Feedback inhibition is the inhibition of an enzyme by the end product of the pathway. The rate of reaction catalyzed by ATCase is fast at low concentrations of CTP but slow as CTP concentration increases. Thus, it continues to make new pyrimidines until sufficient quantities of CTP have accumulated. Feedback inhibition by CTP ensures that N-carbamoylaspartate and subsequent intermediates in the pathway are not needlessly formed when pyrimidines are abundant.

Draw the general chemical formula of a fatty acid and be able to use standard notation for representing the number of carbons and double bonds in a fatty acid chain.

For example, C18 is called octadecanoic acid because the parent hydrocarbon is octadecane with no double bonds (2=octadecadienoic acid, etc). The notation 18:0 denotes a C18 fatty acid with no double bonds.

Describe the structures and biological roles of glycogen, starch, amylose, amylopectin, and cellulose.

Glycogen is the storage form of glucose. It is present in most of our tissues but is most abundant in muscle and liver. Glycogen is a large, branched polymer of glucose residues that are mostly linked by alpha-1,4-glycosidic bonds. The nutritional reservoir in plants is the homopolymer starch. There are two forms: amylose and amylopectin. Amylose is the unbranched type of starch consisting of glucose residues in alpha-1,4 linkages. Amylopectin is the branched form that has about 1 alpha-1,6 linkage per 30 alpha-1,4 linkage. More than half the carbohydrate ingested by human beings is starch.

Describe the composition of glycolipids. Note the location of the carbohydrate components of membranes.

Glycolipids are sugar containing lipids. They are derived from sphingosines. The amino group of the sphingosine is acylated by a fatty acid. They differ from sphingomyelin in the identity of the unit that is linked to the primary hydroxyl group of the sphingosine backbone. In glycolipids, one or more sugars are attached to this group.

Explain why the influenza virus would have two proteins, hemagglutinin and neuraminidase, which perform diametrically opposite tasks.

Influenza virus recognizes sialic acid residues linked to galactose residues that are present on cell-surface glycoproteins. The viral protein that binds to these sugars is called hemagglutinin. After binding hemagglutinin, the virus has to exit the cell, a process essentially the reverse of viral entry. Upon complete assembly, the viral particle is still attached to sialic acid residues of the cell membrane by hemagglutinin on the surface of the new virions. Another viral protein, neuraminidase, cleaves the glycosidic bonds between the sialic acid residues and the rest of the cellular glycoprotein, freeing the virus to infect new cells and spreading the infection.

Define lipid and list the major kinds of membrane lipids.

Lipids are water-insoluble biomolecules that are highly soluble in organic solvents such as chloroform. They have a variety of biological roles: fuel molecules, highly concentrated energy stores, signal molecules and messengers in signal-transduction pathways, and components of membranes. The three major kinds of membrane lipids are phospholipids, glycolipids, and cholesterol.

Describe the properties of an amphipathic molecule.

Membrane formation is a consequence of the amphipathic nature of the molecules. Their polar head groups favor contact with water, whereas their hydrocarbon tails interact with one another in preference to water. The polar head groups form the outside surface of a micelle (globular structure), which is surrounded by water, and the hydrocarbon tails are sequestered inside, interacting with one another.

Explain the role of O-glycosidic bonds in the formation of monosaccharide derivative, disaccharides, and polysaccharides.

Oligosaccharides are built by the linkage of two or more monosaccharides by O-glycosidic bonds.

Outline the structural effects of binding of CTP and PALA to ATCase.

PALA is a potent competitive inhibitor of ATCase that binds to and blocks the active sites. The structure of the ATCase-PALA complex reveals that PALA binds at sites lying at the boundaries between pairs of catalytic chains within a catalytic trimer. Each catalytic trimer contributes three active sites to the complete enzyme. It also reveals a remarkable change in quaternary structure. The two catalytic trimers move 12 A farther apart and rotate 10 degrees about their common threefold axis of symmetry. The regulatory dimers rotate 15 degrees to accommodate this motion. The enzyme literally expands from PALA. Therefore, there are two quaternary forms: one that predominates in the absence of substrate or substrate analogs (Tense state) and one that predominates when substrates or analogs are bound (Relaxed state). The binding of CTP to the T state shifts equilibrium to favor the T state, decreasing net enzyme activity.

Describe the reaction catalyzed by aspartate transcarbamoylase (ATCase), the regulation of ATCase by CTP and ATP, and the biological significance of this regulation.

Reaction: ATCase catalyzes the first step in the biosynthesis of pyrimidines: the condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate. This reaction is the committed step in the pathways and consists of 10 reactions that yield the pyrimidine nucleotides uridine triphosphate (UTP) and cytidine triphosphate (CTP). Regulation: ATCase is inhibited by CTP, the final product of the ATCase-initiated pathway. The inhibitory ability of CTP is remarkable because it is structurally quite different from the substrates of the reaction. Thus, it must bind to a site distinct from the active site at which substrate binds (allosteric or regulatory site). It is an example of an allosteric inhibitor. In ATCase, the catalytic and regulatory sites are on separate polypeptide chains. ATP is also an example, and it binds to the same site as CTP. However, ATP binding stabilizes the R state which increases the reaction rate. At high concentration of ATP, kinetic profile shows a less-pronounced sigmoidal behavior because this prevents CTP from inhibiting the enzyme (bind at same site).

Explain how trypsin is inhibited by the pancreatic trypsin inhibitor.

Specific protease inhibitors terminate proteolysis; an example is the pancreatic trypsin inhibitor. It inhibits trypsin by binding very tightly to its active site. Pancreatic trypsin inhibitor is a very effective substrate analog. In addition, there are many hydrogen bonds between the main chain of trypsin and that of its inhibitor. The carbonyl group of lysine 15 and the surrounding atoms of the inhibitor fit snugly in the active site. The structure of the enzyme is not change by binding of the inhibitor, so it is preorganized into a structure that is highly complementary to the active site.

Name the amino acid residues that are used for attachment of carbohydrates to glycoproteins.

Sugars in glycoproteins are attached either to the amide nitrogen atom in the side chain of asparagine or to the oxygen atom in the side chain of serine or threonine.

Explain what makes a sugar a reducing sugar.

Sugars that react are called reducing sugars. Reducing sugars can often nonspecifically react with a free amino group to form a stable covalent bond.

List the main roles of carbohydrates in nature.

The cells of all organisms are coated in a dense and complex coat of carbohydrates. The environment in which cells live is rich in secreted carbohydrates central to cell survival and cell-to-cell communication. Carbohydrates are required for interactions that allow cells to form tissues, are the basis of human blood groups, and are used by a variety of pathogens to gain access to their hosts. Indeed, rather than being mere infrastructure components, carbohydrates supply details and enhancements to the biochemical architecture of the cell, helping to define the functionality and uniqueness of the cell.

Explain how ring structures arise through the formation of hemiacetal and hemiketal bonds.

The chemical basis for ring formation is that an aldehyde can react with an alcohol to form a hemiacetal. Similarly, a ketone can react with an alcohol to form a hemiketal.

Differentiate between concerted and sequential mechanisms of allosteric regulations and describe the experimental evidence for a concerted allosteric transition during the binding of substrate analogs to ATCase.

The concerted model of allosteric regulation causes a change in the enzyme that is "all or none." The entire enzyme is converted from T into R, affecting all of the catalytic sites equally. In the sequential model, it assumes that all the binding of ligand to one site on the complex can affect neighboring sites without causing all subunits to undergo the T to R transition. The concerted model explains the behavior of ATCase well. Much like an "on and off" switch, cooperativity ensures that most of the enzyme is either on (R state) or off (T state).

List the common covalent modifications used to regulate protein activity.

The covalent attachment of a molecule to an enzyme or protein can modify its activity. Most modifications are reversible. Phosphorylation and dephosphorylation are common means of covalent modification. The attachment of acetyl groups to lysine residues by acetyltransferases and their removal by deacetylases are another example. Histones are extensively acetylated and deacetylated in vivo on lysine residues. Protein acetylation appears to be especially important in the regulation of metabolism. An example of a case where modification is not reversible is the attachment of a lipid group which causes some proteins in signal transduction pathways to become affixed to the cytoplasmic face of the plasma membrane. This allows the protein to be able to receive and transmit information that is being passed along their signaling pathways.

Explain the purpose of isozymes in metabolism.

The existence of isozymes permits the fine-tuning of metabolism to meet the needs of a given tissue or developmental stage. Human beings have two isozyme polypeptide chains for this enzyme: the H isozyme is highly expressed in heart muscle and the M isozyme is expressed in skeletal muscle. There can be many different combinations of the two. The H4 isozyme has a higher affinity for substrate than M4. High levels of pyruvate allosterically inhibit H4 but not M4. M4 functions optimally in the anaerobic environment of hard-working skeletal muscle whereas H4 does so in the aerobic environment of heart muscle.

Define zymogen. Give examples of enzymes and proteins that are derived from zymogens and the biological processes they mediate.

The inactive precursor of an enzyme is called a zymogen. 1. Digestive enzymes that hydrolyze foodstuffs are synthesized as zymogens in the stomach and pancreas. 2. Blood clotting is mediated by a cascade of proteolytic activations that ensures a rapid and amplified response to trauma. 3. Some protein hormones are synthesized as inactive precursors. Insulin is derived from proinsulin by proteolytic removal of a peptide. 4. Collagen, the major constituent of skin and bone, is derived from procollagen, a soluble precursor. 5. Many developmental processes are controlled by the activation of zymogens. 6. Programmed cell death or apoptosis is mediated by proteolytic enzymes called caspases, which are synthesized in precursor form as procaspases. When activated, caspases function to cause cell death.

Outline the effects of heterotropic and homotropic allosteric interactions on the equilibrium between the T and R forms of ATCase.

The presence of additional substrate increases the fraction of enzyme molecules in the more active R state because the position of equilibrium depends on the number of active sites that are occupied by substrate. The effects of substrates on allosteric enzymes are referred to as homotropic effects. The effects of nonsubstrate molecules on allosteric enzymes are referred to as heterotropic effects. Substrates generate the sigmoidal curve while regulators shift the KM.

Identify similarities between cooperativity in ATCase and hemoglobin.

The vast majority of allosteric enzymes display sigmoidal kinetics like hemoglobin. Also, allosteric enzymes like ATCase demonstrate cooperativity between subunits. Lastly, ATCase also displays a T state and an R state depending on specific substrate bound.

Summarize the enzymes and conditions required for the activation of all the digestive enzymes.

The zymogens must be switched on at the same time. Coordinated control is achieved by the action of trypsin as the common activator of all pancreatic zymogens—trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, and prolipase.

Explain the role of thrombin in the activation of fibrinogen into fibrin and describe the structure of fibrin arrays.

Thrombin is synthesized as a zymogen called prothrombin. Fibrinogen is made up of three globular units connected by two rods. Thrombin cleaves four arginine-glycine peptide bonds in the central globular region of fibrinogen. On cleavage, an A peptide of 18 residues is released from each of the two A-alpha chains, as is a B peptide of 20 residues from each of the two B-beta chains. These A and B peptides are called fibrinopeptides. Peptide bond cleavage exposes new amino termini that can participate in specific interactions. The newly formed "soft clot" is stabilized by the formation of amide bonds between the side chains of lysine and glutamine residues in different monomers.