Biochem exam 2

explain how the structure of hemoglobin changes on oxygen binding.

R state: When O binds to the iron in the heme group, the iron atom is pulled into the plane of porphoryn ring. causes structural changes → salt bridges and H bonds broken, higher O affinity.

identify the key regulators of hemoglobin function.

Allosteric effectors: 2,3 BPG, CO2, H+. All stabilize T state and influence oxygen binding affinity. CO is a competitive inhibitor but not an allosteric effector.

define the Bohr effect and describe how it explains maximal oxygen delivery to tissues in greatest need.

rapidly metabolizing tissues produce CO2 → blood pH lowers, favors T state → oxygen release promoted in tissues → protonation of His146 increases, forming salt bridges that stabilize T state → facilitates CO2 transport

give several examples of hemoglobin mutations that result in disease.

sickle cell disease (single sub glu → val, creates hydrophobic contact point, hydrophobic aa become exposed in T state, causes aggregation), α-thalassemia (decreased production of α-chains, less β-chains which bind to O2 better), β-thalassemia (decreased production of β-chains, α-chains form insoluble aggregates.)

explain the difference between the free energy change of a reaction and the standard free energy change, and use the relationship between the two to determine ∆G, ∆G˚

free energy change: ∆G. Standard free energy change: ∆G˚ (∆G under standard conditions of 1M, 298k, 1atm, pH7)

describe how catalysts affect the rate of a reaction

catalysts lower activation energy and stabilize the transition state(s), do not affect ∆G or keq.

draw free energy diagrams to represent both exergonic and endergonic reactions in the presence and absence of a catalyst

explain the relationship between the transition state and the active site of an enzyme, and list several characteristics of active sites.

the active site is specific to one substrate, binds the transition state tighter than the substrate, lowering activation energy, changes shape upon substrate binding (induced fit)

define reaction velocity

the initial rate of catalysis at initial substrate concentration

explain how reaction velocity is measured, and how reaction velocities are used to characterize enzyme activity

V₀= k₁[s]. at constant [e] increasing [s] increases rate until saturation. at constant [s] increasing [e] increases rate.

describe the Michaelis-Menten model and explain the assumptions used to derive the Michaelis-Menten equation

steady state assumption. [s] lowers, [p] increases, [e] and [es] stay steady.

differentiate between the Michaelis-Menten parameters ν_max, k_cat, and K_M

k_cat: turnover rate (number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is fully saturated with substrate.) K_M: michaelis constant (measure of affinity. represents [s] at which reaction rate ν_max/2, low K_M indicates high affinity)

evaluate the efficiency and specificity of an enzyme based on its kinetic parameters

describe the characteristics of allosteric enzymes as well as the shape of their reaction velocity vs. substrate concentration curves

regulated by allosteric effectors that bind to sites other than the active site. have a steep slope near K_M.

define feedback inhibition

a cellular control mechanism, in which the activity of an enzyme is inhibited by the end product of a biochemical pathway

describe the regulatory mechanism used by aspartate transcarbamoylase (ATCase)

uses feedback inhibition (allosteric regulation). end product CTP inhibits ATCase, ATP activates it. ATCase exists in T and R state. CTP binds to the T state, stabilizes it and decreases activity (K_M increases). ATP binds to R state, stabilizing it and increasing activity (K_M decreases).

differentiate between homotropic and heterotropic effects and explain how they modify the equilibrium between the T and R forms of an allosteric enzyme.

Homotropic: substrate is the effector. binding of the substrate to one active site increases the substrate affinity and catalytic activity of other active sites on the same enzyme. typically shift equilibrium towards R state.

Heterotropic: the effector is a molecule other than the substrate. binding of the effector to a regulatory site alters the enzymes conformation and activity. can shift equilibrium towards T state (inhibitors) or R state (activators).

identify common strategies used by enzymes to accelerate reaction rates

approximation and orientation: enzymes bring substrates together in the correct orientation within the active site, increases the likelihood of productive collisions.

transition state stabilization: enzymes bind the transition state more tightly than they bind the substrate, lowers activation energy.

covalent catalysis: enzyme forms temporary covalent bond with the substrate.

general acid-base catalysis: aa side chains in active site serve as acids or bases to facilitate reaction.

metal ion catalysis: can stabilize charges, orient substrates, or directly participate in redox reactions.

describe how temperature and pH can affect the rate of an enzyme reaction

temp: more collisions at higher temps, until temp is too high and enzyme denatures. pH: bell shaped curve, asp/glu/c term co2- needs to be deprotonated for max activity, lys/N term NH3+ needs to be protonated for max activity.

explain the mechanism of catalysis by chymotrypsin

describe how inhibitors can be used to study enzyme mechanisms

Irreversible Inhibitors: Form covalent bonds, permanently blocking activity. Help identify essential active site residues.

• Group-Specific Reagents: React with specific amino acid side chains. Example from the notes: DIPF helped to identify the key serine in chymotrypsin.

• Affinity Labels: Resemble the substrate and covalently bind in the active site. TPCK, mentioned in the notes, is an affinity label for chymotrypsin that helped determine the importance of histidine 57.

• Reversible Inhibitors: Bind non-covalently, allowing for study of how inhibition affects Vmax and KM.

  • Competitive: Compete with substrate for the active site. Often resemble the substrate or transition state. Transition state analogs, particularly effective competitive inhibitors, support the role of transition state stabilization in catalysis.

  • Uncompetitive: Bind only to the enzyme-substrate complex.

  • Non-competitive (Mixed): Bind to both enzyme and enzyme-substrate complex. Can reveal allosteric sites.

distinguish between reversible and irreversible inhibitors

irreversible: form E-I covalent bonds, reversible: form non-covalent bonds/interactions

describe how competitive, uncompetitive, and noncompetitive inhibitors alter the kinetics of an enzymatic reaction.

explain the difference between an aldose & a ketose

aldose has an aldehyde group, ketose has a ketone group

identify the anomeric carbon in any monosaccharide and the reducing end of any polysaccharide

anomeric carbon: stereocenter formed when ring closes, attached to to O’s. Reducing end: has 1 free OH at anomeric C. ring exists in equilibrium with the open chain form.

draw the open chain and cyclic forms of D-glucopyranose as well as the cyclic form of D-ribofuranose

recognize the stereochemical relationships between epimers and anomers

describe the glycoside bond connecting two monosaccharides in a di- or polysaccharide

describe the three main classes of glycoconjugates and explain their biological roles.

glycoproteins: aids in folding, cell surface recognition, prevents aggregation.

glycolipids: cell membrane structure, cell signaling

proteoglycans: proteins attached to GAGS. lubricants, cell-cell adhesion, binds to proteins that regulate proliferation.