Biochem Week 3 Flashcards
(39 cards)
Define rate constant of a ligand protein interaction, and correlate rate constants to Ka and Kd
Kd is the dissociation constant given by the equation Kd=k2/k1 . Small k2 compared to k1 favors reaction going forward (enzyme-substrate, or ligand binding formation) and greater affinity of enzyme with substrate.
Describe how measurable parameters can be used to determine the initial velocity of an enzyme catalyzed reaction using the Michaelis-Menten equation
Vo=k3[ES] gives initial velocity of the reaction but [ES] cannot be measured so instead use the idea that at a saturating concentration of substrate Vo=Vmax because at this condition [Et]=[ES] therefore Vmax=k3[Et]
Define Km, and describe its practical implication in understanding the efficiency of an enzyme-catalyzed reaction
Km=S when Vo=Vmax/2 Km of the enzyme for a substrate is the concentration of substrate required to reach 1/2*Vmax Km is a measure of the apparent affinity of the enzyme for its substrate. Lower Km means substrate has a higher affinity for the enzyme.
Deduce the Michaelis-Menten equation from the ligand-binding equation
Ligand-Binding equation: B = (Bmax[H])/(Kd+[H]) Michaelis-Menten equation: Vo = (Vmax[S])/(Km+[S])
Distinguish between ligand binding and enzyme catalysis, and describe the relationship between Km and Kd
Ligand binding (ex. Hormone-Receptor) and enzyme catalysis can be described in a similar manner mathematically because both have two reactants favoring a single product. Km and Kd are both ways of showing the favorability of a reaction. Km and Kd are the same when the dissociation of Enzyme-Substrate to Enzyme and Product is ignored.
Define Vmax, and explain what is meant by saturation kinetics
The approach to the finite limit of Vmax is called saturation kinetics because velocity cannot increase any further once the enzyme is saturated with substrate.
Explain the mechanistic significance of the hyperbolic nature of the Michaelis-Menten curve
According to the curve Vmax will only be reached at an infinite concentration of substrate, which leads to the idea of saturation kinetics.
Define isozymes, and explain the physiological significance of different isoforms having different Km’s for substrate
Isozymes are enzymes that catalyze the same reaction but have different primary or quaternary structures, which permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage. Isozymes generally have different kinetic and regulatory properties, cofactors, and subcellular or tissue distribution
Explain hexokinase and glucokinase as they pertain to isozymes
Hexokinase (in the brain) and Glucokinase (in hepatocytes). Hexokinase has low Km and low Vmax for glucose. Glucokinase has higher Km and very high Vmax for glucose. The glucose sparing effect of the liver allows the brain to get the glucose it needs before the liver begins to store glucose (bad things would happen if liver stored glucose before the brain got what it needed).
Explain what is meant by reversible inhibition of enzymes, and describe the mechanistic difference between reversible and irreversible inhibitions
Reversible inhibition of enzymes means something other than the substrate binds to the enzyme limiting its effectiveness in producing product. The inhibition is reversible because the inhibitor can leave and the enzyme can return to its effectiveness. Irreversible inhibitors the enzyme will never be able to take part in the reaction to turn substrate into product. This drastically reduces Vmax of the reaction taking place.
Explain the Lineweaver-Burke (double reciprocal) transformation of the Michaelis-Menten equation
Enzymes obeying michaelis-menten relationship can be plotted as a straight line by plotting 1/VO vs 1/[S]. The Lineweaver-Burke (double reciprocal) transformation provides: a more accurate determination of Vmax and Km, better distinguishment among certain types of enzymatic reactions, and allows for analysis of enzyme inhibition.
Distinguish between competitive, noncompetitive and uncompetitive reversible inhibition
Competitive inhibitor binds to the same active site as the substrate, preventing substrate from binding to enzyme. Noncompetitive inhibitor binds to a different active site than the substrate but its binding essentially lowers the concentration of enzyme available for product formation. According to Marks text, “Uncompetitive inhibition is almost never encountered in medicine and will not be discussed further.” Dr. Dey’s definition of uncompetitive inhibition is when an enzyme has two active binding sites A and B. Substrate A is allowed to bind but Substrate B cannot bind because an uncompetitive inhibitor has bound to site B, thus rendering the enzyme inactive (from the perspective of substrate B this could be classified as competitive inhibition).
Demonstrate how the double reciprocal plot can be used to discriminate one mechanism (competitive and non-competitive inhibition) from the other
Competitive inhibitor rotates slope of double reciprocal plot line along the 1/V axis (slope changes, 1/V intercept doesn’t change). - Lines meet on Y axis (Vmax doesn’t change, Km is increased) Noncompetitive inhibitor rotates slope of double reciprocal plot line along the 1/[S] (slope changes, 1/[S] intercept doesn’t change). - lines meet on X axis (Km (affinity) doesn’t change, Vmax is decreased) Uncompetitive inhibitor changes the 1/V and 1/[S] slope intercepts but not the slope itself.
Define/describe allosteric regulation of enzymes:
Allosteric regulation of enzymes: • Regulation by reversible noncovalent binding of regulatory compounds • The regulatory compounds are called allosteric modulators • Modulators induce conformational changes switching between more active and less active forms • Modulators could be inhibitory or stimulatory
What is the T and R states of an allosteric enzyme?
Structurally distinct enzyme forms that occur after conformational change between a low-activity, low-affinity “tense” or T state and a high-activity, high-affinity “relaxed” or R state. Also can thought of as the first conformation = T state; the second = R state. • Higher concentrations of substrate favor the conversion of the T state to the R state.
Distinguish between homotropic and heterotropic allosteric regulation
Homotropic: A homotropic allosteric modulator is a substrate for its target enzyme, as well as a regulatory molecule of the enzyme’s activity. It is typically an activator of the enzyme. • Example: O2 is a homotropic allosteric modulator of hemoglobin. Heterotropic: A heterotropic allosteric modulator is a regulatory molecule that is not also the enzyme’s substrate. It may be either an activator or an inhibitor of the enzyme. • Example: H+, CO2, and2,3-bisphosphoglycerate are heterotropic allosteric modulators of hemoglobin.
Describe cooperativity in substrate binding to multisubunit protein with reference to hemoglobin
Cooperativity is a phenomenon displayed by systems involving identical or near-identical elements, which act non-independently of each other, relative to a hypothetical standard non-interacting system in which the individual elements are acting independently. One manifestation of this are enzymes or receptors that have multiple binding sites where the affinity of the binding sites for a ligand is apparently increased, positive cooperativity, or decreased, negative cooperativity, upon the binding of a ligand to a binding site. • Example: The affinity of hemoglobin’s four binding sites for oxygen is increased above that of the unbound hemoglobin when the first oxygen molecule binds.
Distinguish the action of allosteric activators and inhibitors by inspection of a V0/ Vmax versus [S] plot
Allosteric inhibition: occurs when the binding of one ligand decreases the affinity for substrate at other active sites. For example, when 2,3-BPG binds to an allosteric site on hemoglobin, the affinity for oxygen of all subunits decreases. This is when a regulator is absent from the binding site. Allosteric activation: occurs when the binding of one ligand enhances the attraction between substrate molecules and other binding sites. An example is the binding of oxygen molecules to hemoglobin, where oxygen is effectively both the substrate and the effector. The allosteric, or “other”, site is the active site of an adjoining protein subunit. The binding of oxygen to one subunit induces a conformational change in that subunit that interacts with the remaining active sites to enhance their oxygen affinity.
How do covalent modifications regulate enzymes?
Enzyme activity may increase or decrease after the covalent addition of a chemical group. 1. Phosphorylation affects many enzymes. a. Pyruvate dehydrogenase and glycogen synthase are inhibited by phosphorylation. b. Glycogen phosphorylase is activated by phosphorylation. 2. Phosphatases that remove the phosphate groups alter the activities of these enzymes. 3. Phosphorylation introduces negative charges to the protein, which may alter the secondary and tertiary structures.
How does the protein-protein interaction with calmodulin regulate an enzyme?
Calmodulin (CaM) (an abbreviation for CALcium-MODULated proteIN) is a calcium-binding messenger protein expressed in all eukaryotic cells. • Calmodulin induces substantial conformational change in the protein • The calcium-induced conformational change exposes hydrophobic surfaces in calmodulin to mediate interaction with other proteins and subsequently modulate their function
What are the major differences in Hb and Mb
Differences in Hb and Mb - Mb is a storage protein – binds O2 avidly, dissociates slowly - Mb is not cooperative - Mb is 1 polypeptide vs Hb is multiple subunits = tertiary structure
Compare and contrast myoglobin and hemoglobin structure:
II. MYOGLOBIN: an oxygen carrier in muscle cells A. Physico-chemical properties i. 153 amino acids – single polypeptide chain ii. Very compact: globular structure →little empty space for solution to get in iii. Tertiary structure: 8 alpha helices (A-H), 4 helices terminated by proline residues iv. About 75% is in alpha helical structure v. Polar side chains on outside of protein →interact with solution vi. Myoglobin = storage protein →mainly in skeletal muscle vii. High O2 affinity – does not change with concentration viii. Monomer →no cooperativity Myoglobin globin fold A single polypeptide chain. Built primarily of a-helices and turns Presence of heme (prosthetic group) allows reversible binding of oxygen Heme consists a protoporphyrin and a central iron atom Interior consists almost entirely of hydrophobic residues The only polar residues inside are the two Histidines that play critical role in binding of heme oxygen Interaction of O2 with heme is often accompanied by conformational changes Protein structure affects how ligands bind Bound O2 is hydrogen bonded to a Histidine (His E7) residue Myoglobin and b-Subunit of Hemoglobin Have Structurally Related Globin Folds III. HEMOGLOBIN A. Structure i. Primary, secondary and tertiary structures are same as Mb ii. More than 1 subunit →quaternary structure: A tetrameric Protein with four heme prosthetic groups Adult hemoglobin contains two a- and two b- chains Strong interaction between unlike subunits Hydrophobic interaction predominates Hemoglobin is an oxygen carrier in blood Binding of oxygen induces a conformational change in hemoglobin which is transmitted to other subunits iv. Geometric explanation 1. Hemes are dome-shaped 2. O2 binds →pulls Fe down →dome becomes flat →pulls helix
Interpret the saturation curve of oxygen binding by hemoglobin and myoglobin at increasing partial pressure of O2, and explain the physiological significance of cooperativity in O2 binding to hemoglobin
As demonstrated by the graph a single subunit has very low affinity even as pO2 increases and only reaches about 50% saturation. Contrast this to the rapid rise of hemoglobin, once activated from the T to the R state it quickly is able to bind O2 easily.
Describe the role of heme in oxygen binding by hemoglobin and illustrate why free heme cannot substitute for hemoglobin in transporting oxygen
Heme improves affinity of oxygen in the hemoglobin molecule, example of cooperativity. When oxygen binds to the iron complex, it causes the iron atom to move back toward the center of the plane of the porphyrin ring. At the same time, the imidazole side-chain of the histidine residue interacting at the other pole of the iron is pulled toward the porphyrin ring. This interaction forces the plane of the ring sideways toward the outside of the tetramer, and also induces a strain in the protein helix containing the histidine as it moves nearer to the iron atom. This strain is transmitted to the remaining three monomers in the tetramer, where it induces a similar conformational change in the other heme sites such that binding of oxygen to these sites becomes easier.
