Principles of Biochemistry Lecture 19a PDF, Spring 2024, Weill Cornell Medicine-Qatar

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 19a & 19b Regulation of carbohydrates metabolism Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 13: p. 496-503 19a/ GENERAL PRINCIPLES OF METABOLIC REGULATION HOMEOSTASIS Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state In steady state, the rate of synthesis of a metabolite equals the rate of breakdown of that metabolite The flux (or flow of metabolites) through the pathways is regulated to maintain homeostasis Pathways are at steady state unless perturbed After perturbation, a NEW steady state will be established FEEDBACK INHIBITION In many cases, ultimate products of metabolic pathways directly or indirectly inhibit their own biosynthetic pathways – ATP inhibits the committed step of glycolysis PFK-1 ATP RATES OF BIOCHEMICAL REACTIONS Rates of biochemical reactions depend on many factors: Concentration of reactants vs. products Activity of the catalyst – Concentration of the enzyme – Rate of translation (synthesis) vs. rate of degradation – Intrinsic activity of the enzyme – Could depend on substrate, effectors or phosphorylation state Concentrations of effectors – Allosteric regulators – Competing substrates – pH, ionic environment Temperature FACTORS AFFECTING THE ACTIVITY OF ENZYMES The total activity of an enzyme can be changed by: Altering the number of its molecules in the cell, or Altering its effective activity in a subcellular compartment (1 through 6), or Modulating the activity of existing molecules (7 through 10 ). An enzyme may be influenced by a combination of such factors. Km VS. METABOLITE CONCENTRATION RATE OF REACTION DEPENDS ON THE CONCENTRATION OF SUBSTRATES The rate is more sensitive to substrate concentration below the Km – Frequency of substrate meeting the enzyme matters The rate becomes insensitive at high substrate concentrations (above the Km) – The enzyme is nearly saturated with substrate Example: Effect of ATP concentration on the initial reaction velocity of a typical ATPdependent enzyme. These experimental data yield a Km for ATP of 5 mM. The concentration of ATP in animal tissues is ~5 mM (close to the Km). Km VS. [METABOLITE] ELASTICITY COEFFICIENT MEASURES THE RESPONSIVENESS TO SUBSTRATE Elasticity coefficient, ε, of an enzyme with typical Michaelis-Menten kinetics. u At [S] > Km, increasing [S] has little effect on V; ε here is close to 0. (the enzyme works at the Vmax and is not responsive to [S], because it is already saturated) ALLOSTERIC EFFECTORS CAN INCREASE OR DECREASE ENZYME ACTIVITY typically convert hyperbolic kinetics to sigmoid kinetics cooperativity of an allosteric protein can be expressed as a Hill coefficient, with higher coefficients meaning greater cooperativity Relationship between Hill Coefficient and the Effect of Substrate Concentration on Reaction Rate for Allosteric Enzymes Hill coefficient (nH) 0.5 Michaelian enzyme 1.0 Required change in [S] to increase V0 from 10% to 90% Vmax × 6,600 × 81 2.0 ×9 3.0 × 4.3 Allosteric enzyme 4.0 ×3 PHOSPHORYLATION OF ENZYMES AFFECTS THEIR ACTIVITY Phosphorylation is catalyzed by protein kinases Dephosphorylation is catalyzed by protein phosphatases, or can be spontaneous Typically, proteins are phosphorylated on the hydroxyl groups of Ser, Thr or Tyr ENZYMES ARE ALSO REGULATED BY REGULATORY PROTEINS Binding of regulatory protein subunits affects specificity Example: Structure and action of phosphoprotein phosphatase 2A (PP2A). PP2A recognizes several target proteins, its specificity provided by the regulatory subunit. Each of several regulatory subunits fits the scaffold containing the catalytic subunit, and each regulatory subunit creates its unique substrate binding site. PROTEINS HAVE A FINITE LIFESPAN Different proteins in the same tissue have very different half-lives – Less than an hour to about a week for liver enzymes – Stability correlates with the sequence at N-terminus (Nend rule) Some proteins are as old as you are – Crystallins in the eye lens Turnover = synthesis followed by degradation rapid turnover is energetically expensive, but proteins with a short half-life reach new steadystate levels faster than those with a long half-life REACTIONS FAR FROM EQUILIBRIUM ARE COMMON POINTS OF REGULATION Within a metabolic pathway most reactions operate near equilibrium Key enzymes operate far from equilibrium – These are the sites of regulation – Control flow of metabolites through the pathway To maintain steady state all enzymes operate at the same net rate (but different rates of forward vs reverse reactions) far from equilibrium near equilibrium Near-equilibrium and non-equilibrium steps in a metabolic pathway. Steps 2 and 3 of this pathway are near equilibrium in the cell; for each step, the rate (V) of the forward reaction is only slightly greater than the reverse rate, so the net forward rate (10) is relatively low and the free-energy change, ∆G, is close to zero. An increase in [C] or [D] can reverse the direction of these steps. Step 1 is maintained in the cell far from equilibrium; its forward rate greatly exceeds its reverse rate. The net rate of step 1 (10) is much larger than the reverse rate (0.01) and is identical to the net rates of steps 2 and 3 when the pathway is operating in the steady state. Step 1 has a large, negative ∆G. NEAR EQUILIBRIUM vs. NON EQUILIBRIUM REACTIONS (Keq vs. Q) § Reaction: A+BàC+D § At equilibrium à Keq § In the cell à Q § Q=[C][D]/[A][B] To identify near equilibrium reactions in a cell, the mass action ratio Q must be compared to the equilibrium constant for the reaction Keq: - When Q and Keq are within 1 or 2 orders of magnitude of each others à the reaction is near-equilibrium (6 of the 10 steps of glycolysis) - When Q and Keq are much more than 1 or 2 orders of magnitude of each others à the reaction is far from equilibrium (3 regulated steps of glycolysis) ATP AND AMP ARE KEY CELLULAR REGULATORS A 10% decrease in [ATP] can greatly affect the activity of ATP utilizing enzymes A 10% decrease in [ATP] leads to a dramatic increase in [AMP] – AMP can be a more potent allosteric regulator * In some processes, ADP is converted in the cell to AMP by adenylate kinase: 2ADP à ATP +AMP AMP DIFFERENTIALLY AFFECTS PATHWAYS IN DIFFERENT TISSUES VIA AMPK Role of AMP-activated protein kinase (AMPK) in carbohydrate and fat metabolism: AMPK is activated by elevated [AMP] or decreased [ATP], by exercise, by the Sympathetic Nervous System (fight or flight response), or by peptide hormones leptin and adiponectin produced in adipose tissue. When activated, AMPK phosphorylates target proteins and: shifts metabolism in a variety of tissues away from energy-consuming processes such as the synthesis of glycogen, fatty acids, and cholesterol; shifts metabolism in extra-hepatic tissues to the use of fatty acids as a fuel; and triggers gluconeogenesis in the liver to provide glucose for the brain. In the hypothalamus, AMPK stimulates feeding behavior to provide more dietary fuel. SOME ENZYMES IN THE PATHWAY LIMIT THE FLUX OF METABOLITES MORE THAN OTHERS Enzymes that catalyze reactions far from equilibrium (regulated) Not all regulated enzymes have the same affect on the entire pathway – Some control flux (metabolite flow) through the pathway – Others regulate steady state concentrations of metabolites in response to changes in flux Examples: Hexokinase and phosphofructokinase are appropriate targets for regulation of glycolytic flux – Increased hexokinase activity enables activation of glucose – Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis HEXOKINASE AFFECTS FLUX THROUGH GLYCOLYSIS MORE THAN PHOSPHOFRUCTOKINASE Dependence of glycolytic flux in a rat liver homogenate on added enzymes. Purified enzymes in the amounts shown on the x axis were added to an extract of liver carrying out glycolysis in vitro. The flux through the glycolytic pathway is shown on the y axis. FLUX TO GLYCOGEN SYNTHESIS IS CONTROLLED BY GLUCOSE UPTAKE AND PHOSPHORYLATION Control of glycogen synthesis from blood glucose in muscle. Transport Insulin affects 3 of the 5 steps in this pathway, but it is the effects on transport and hexokinase activity, not the change in glycogen synthase activity, that increase the flux (flow of metabolites) toward glycogen. Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 14: p. 539-546