5 - Enzymes 2 Flashcards
Describe the Michaelis-Menten theory? What situations does it model?
Enzymes
The Michaelis Menten Model is valid only when [E]<[S].
S binds to E forming an ES intermediate. The ES intermediate decomposes to E+P. E, S, and ES are in equilibrium. Formation of P from ES is the rate limiting steo in the reaction (nonreversible, but the reverse reaction can be catalyzed by a different enzyme). Kcat, turnover constant, signifies the catalytic efficienty of the enzyme.
Describe how a graph of Michaelis-Menten kinetics can be understood.
Enzymes
- Initial linear portion follows first order kinetics: [S]↑∝V↑
- Further [S]↑ begins to saturate the solution as ES→E+P is the rate limiting step.
- at [S]sat we have Vmax and V is independent of any [S]↑, this final portion follows Zeroth order kinetics
- K_m=[S] at 1/2 Vmax
If there were two substrates, the graph would follow second order kinetics initially. Glucokinase and hexokinase catalyze the same reaction, phosphorylation of glucose to glucose-6-phosphate (G-6-P). They exhibit different kinetic properties and have different tissue distributions and physiologic functions.
What is a Lineweaver-Burk plot?
Enzymes
a double-reciprocal plot of the steady-state michaelis-menten equation. SSMM approaches Vmax asymptotically: difficult to extract Vmax and consequently Km.
1. Km: inverse measure of affinity of the enzyme for the
substrate; low K = high affinity
2. Vmax ∝ [E]
3. intersection with the 1/S x-axis = -1/K m
4. intersection with 1/V y-axis = 1/Vmax
Inhibition of an enzyme molecule by a nonphysiologic agent (e.g., drugs/toxins) occurs by complete inactivation of the enzyme. In contrast, inhibition by metabolites, called allosteric inhibitors, occurs by a gradual
reduction in the enzyme activity.
What are the types of inhibition? Describe a situation that exemplifies each type.
Enzymes
- Competitive: structural analogs that compete with substrate for enzyme active site. EI (enzyme-inhibitor) complex formed. Can be overcome by [S]↑ such that [I]«[S]. Km increased, Vmax unchanged. Shift MM graph to right. LB: y-axis intersection same, increase in slope (-1/Km x-axis decreases).
- Noncompetitive: inhibitor deactivates enzyme in a form of allosteric inhibition. Lowers the effective [E], thus also lowers Vmax. Km unchanged as it does not change binding affinitity of original enzyme. Shifts MM graph down. LB: -1/Km x axis same, 1/Vmax intersection goes up (steeper) as Vmax goes down.
- Uncompetitive: reversible inhibition, inhibitor bind to ES complex: ES+I→ESI. Lowers ES, so ES→E+P dissociation is inhibited. DOES NOT bind to free enzyme. Km and Vmax both decreased. Lowers effective [E] so Vmax decreases, and lowers apparent Km since decrease in dissociation ≈ increase in affinity. MM: shifted down and left. LB: same steepness, but 1/Vmax y-axis goes up and -1/Km x axis goes up (more negative, more left). whole graph shifts up.
go back to question to see effect of each inhibitor on lineweaver burke plots
What is an Eadie-Hofstee Plot?
Enzymes
Does not Compress the Data at High Substrate Concentrations
What is an irreversible inhibitor? What is their clinical significance?
Enzymes
- irreversible inhibitors bind to an enzymes active site
- they are substrate analogs->competitive inhibitors
- catalysis generates chemicallly reactive intermediate that inactivates the enzyme by covalent modification
Irreversible Inhibitors Permanently Inactivate Enzymes. The only Means for Reversing the Inhibition is the Synthesis of New Enzyme Protein by the Cell.
Is inhibition equivalent to regulation? Why or why not?
Enzymes
Inhibition, either reversible or irreversible, of an enzyme molecule by a nonphysiologic agent (e.g., drugs and toxins) occurs by complete inactivation of the enzyme. Inhibition is not regulation, which occurs under the influence of normal cellular metabolites
How is enzyme activity regulated in the body? Explain each process.
Enzymes
- Regulation via Reversible Covalent Modification: usually protein kinase phosphorylation. phosphotase can dephosphorylate the regulated enzyme. Phosphorylated state can be active or inactive. Regulation matches the
function of the hormone that generated
the signal (e.g., gluconeogenic enzymes
are turned on by phosphorylation, and
glycolytic enzymes are turned off by
phosphorylation during fasting). - Zymogens: inactive form of enzyme (aka proenzyme). secretion of enzymes in zymogen form prevents autolysis of organs (activate only when cleaved). activation of zymogens involve irreversible covalent modification. many digestive proteolytic enzymes are synthesized like this.
- Allosteric regulation: if an enzyme demonstrates cooperativity: can be influenced by more than one substrate, then allosterism refers to an enzyme’s response to an effector molecule (different substrate) that results in an increase or decrease in its activity. Allosteric effectors induce a conformational change in the enzyme. Positive cooperativity: reaction of a substrate with one active site makes it easier for another substrate to react at another active site. Negative cooperativity: reaction of a substrate with one active site makes it more difficult for a substrate to react at the other active site.
Intracellular signaling pathways occur through a cascade mechanism that is designed to amplify cellular signals. A cascade involves a series of enzymes that sequentially activate each other. Since the product of each activation is a catalyst, each step results in a geometric increase in signal. The last enzyme in the cascade controls a target metabolic pathway or other cellular process. Allosteric inhbition is usually observed in the case of Feedback inhibition.
How do you measure enzyme activity in clinical samples?
Enzymes
not as important
