Biochem Week 3 Flashcards
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.