Enzymes Flashcards
what is the lock and key hypothesis
substrate fits into a substrate binding site to form a reaction intermediate
what is the induced fit hypothesis
enzyme molecule changes shape as the substrate molecule gets close, the change in shape is induced by the substrate binding molecule
what is the significance of the active site
substrate binding site usually close to or equal to active site, the proximity, orientation and chemical environment inside active site as well as helping groups (cofactors/coenzymes) allows chemical reaction to proceed more easily
what occurs if mutation or inhibitor affects substrate binding site?
enzyme has altered affinity for its substrate (increases Km)
what occurs if mutation or inhibitor affects active site?
enzyme will have altered activity (decreased Vmax)
what are the cofactors?
metal ions Fe2+, Mg2+, and Zn2+ ,contribute to catalytic process by acting as electrophiles, assist in the binding of the substrate
what are coenzymes
non-protein organic molecules like vitamins
what is a transition state?
ES in which the bonds are undergoing transformations that are UNLIKE starting material NOR product, usually in a strained or distorted formation, so the energy is increased from the initial state
what does an enzyme do in relation to ES?
enzyme binds to its substrate and reduces the activation energy of the reaction by stabilizing the transition state making the substrate more likely to react and speeds up the reaction
what affects the rate of enzyme catalyzed reactions?
- concentration of enzyme and substrate
- pH
- temp
- activators and inhibitors
what is the relationship between substrate and reaction velocity in a NON-enzymatic reaction?
linear, velocity is proportional to substrate concentration
what occurs when an enzyme catalyzed reaction is at a very high substrate concentration?
velocity increases until Vmax, where enzyme is saturated, and the velocity of the reaction is zero order - constant and independent of S concentration
what occurs when an enzyme catalyzed reaction is at a very low substrate concentration?
the reaction is first order, the velocity is proportional to the concentation of substrate, and velocity increases with respect to substrate concentration
what is drug elimination kinetics?
the metabolism of a drug to inactive metabolites usually by the liver in a first order or zero order elimination kinetic method
how are drugs usually excreted?
kidney, bile duct, lungs, sweat
what is the first order drug elimination kinetic?
a constant FRACTION of drug is eliminated with time, is an exponential decay plot, ie 1/2 is gone in 1 hour, 1/4 in 2 hour, 1/8 in 3 hours, when drug concentration is high, rate of disappearance is also high, the elimination mechanisms are not saturated, a fixed half life
what is the zero order drug elimination kinetic?
constant amount of drug eliminated with time, ie 10mg per hour, no fixed half life, the elimination mechanisms can be saturated
what occurs if high doses of drugs or hepatic/renal diseases impair drug elimination?
elimination process becomes saturated and zero order,
what occurs at extreme pH
proteins are denatured, and active site is distorted and substrate molecules no longer fit in it
what occurs at a slightly different pH than optimal?
alters interaction between E and S, so reaction proceeds at slower rate
what occurs at extreme temperatures?
high and low temps, enzymes denatured and reaction velocity is decreased
what is the michaelis-menten model?
enzyme and substrate combine at the rate of complex formation (k1) to produce ES, then ES proceeds at the rate of product formation (k2) to produce the product E+P
3 assumptions of MM model
- there is a formation of an ES complex
- the concentration of substrate is much greater than [enzyme]
- ES does not change with time (the steady state assumption, initially has a burst and then its concentration will not change demonstrably), and the rate of formation of ES is equal to the rate of breakdown of ES
what is the MM equation?
velocity of product formation= rate of formation of product (k2) x concentration of ES
is equal to velocity = vmax x ([S])/Km + [S]
what is Km
the substrate concentration at half of the max velocity, aka the affinity of the enzyme to substrate, a lower Km indicates a higher affinity, a higher Km indicates a lower affinity
what best describes the MM graph?
it is a combination of zero and 1st order kinetics, where when the S is low, it is 1st order, but when S is high, it is 0 order, aka a hyperbolic curve
what is Kcat?
k2, turnover number, max number of substrate molecules converted to product per enzyme molecule per unit time, =Vmax/enzyme molecule #, units are time^-1
what does a lower Km indicate?
higher affinity and tighter binding, achieves Vmax at low [S]
what does a higher Km indicate?
a lower affinity and weak binding, achieves Vmax at high [S] (takes higher S to achieve Vmax)
what is vmax?
theoretical max velocity of reaction at the saturating substrate concentration
what is the lineweaver-burk double reciprocal plot?
the double reciprocal of MM graph where Km and Vmax can be determined easier
where is Km on a lineweaver Burk plot?
hits X axis, is -1/Km
where is Vmax on lineweaver Burk plot
hits y axis, is 1/vmax
types of enzyme inhibitors
both decrease rate of enzymatic reactions
- irreversible - also called suicide inhibitor or mechanism based inhibitor
- reversible
what do irreversible enzyme inhibitors do?
inhibitor irreversibly inactivates enzyme by destroying or covalently modifying key AA residues - the enzyme CANNOT function anymore, and inhibition cannot be stopped, decreases substrate binding and decreases RR
how does penicillin act as an enzyme inhibitor?
it irreversibly inhibits glycopeptidyl transferase which is required for bacterial cell wall synthesis
how does aspirin act?
irreversibly inactivates COX1 and COX2
what does lead do to the body?
irreversibly inhibits ALA dehydratase and ferrochelatase (needed for heme biosynthesis)
what do organophosphate compounds do to the body?
malathion, parathion, nerve gas sarin, DFP irreversibly inactivate acetylcholinesterase by forming covalent complex with active site serine in Ach-ase
MOA of allopurinol
binds irreversibly to active site of xanthine oxidase, which then converts allopurinol into its active metabolite, oxypurinol, which binds tightly to the active site and is only slowly released
what does xanthine oxidase do?
converts hypoxanthine to xanthine and then to uric acid
2 types of reversible inhibitors
- competitive
2. non competitive
what is a competitive inhibitor?
inhibitor resembles substrate but cannot undergo catalytic step, competes with substrate for binding, SO IT CHANGES THE KM (USUALLY INCREASE), BUT VMAX STAYS THE SAME
example of competitive inhibitor
statin drugs - competitively inhibit rate limiting step in cholesterol synthesis
ex. HMG CoA reductase inhibitor is lipitor, structural analog of HMG CoA
example of competitive inhibitor
methotrexate, structural analog of folic acid and inhibits dihydrofolate reductase and stops thymidine biosynthesis, inhibits DNA replication and mitosis in rapidly growing cells - an antimetabolite
what is a non-competitive inhibitor?
a molecule or ion that binds on a site on th eenzyme so that it affects Vmax - not on substrate binding site, enzyme can bind both substrate and inhibitor, but cannot make a product, decreases kcat and Vmax, no effect on Km
example of non-competitive inhibitor
physostimine, for MG, binds reversibly to Ach-ase away from active site, Km remains the same bc normal binding not affected, but reduces catalytic activity of enzyme, reducing Vmax
example of non-competitive inhibitor
captopril, for HTN, reversible noncompetitive inhibitor of ACE (angiotensin-converting enzyme)
ACE inhibitor mechanism
renin in kidney converts angiotensinogen to angiotensin I, then proteolytically cleaved to make angiotensin II by ACE, angiotensin II increases renal fluid/electrolyte retention
