BICH 410 1st Edition Lecture 20 Outline of Last Lecture I. EnzymesCurrent Lecture- Vmax- value of vmax varies on conc. of enzyme- Specific activity= vmax/g amount of proteino Number of substrate molec converted to product/ amount of tot protein present when Eis saturated with substrateo Specific activity SA increases until total protein present is E (pure protein) at which time it will reach maxo Increase in enzyme E will not increase SA when pure because velocity is proportional to E present- under saturating conditions As long as conditions always the same SA will always be the same As E is doubled activity is doubled therefore once enzyme is pure SA doesn’t change 25mg total protein and 14250umol S to P per sec- SA= VMAX/amount of protein= 14250/25= 579ugmol/secmgPure Pure protein 2mg/ml catalyzes 1456umol/ml S to P/sec- SA= 1456umol/ml S to P/sec divided by 2mg/ml= 728 umol/secmg Calculate percent purity of 1st solno Relationship between SA and Turnover number TO= kcat=vmax/E= SAxMolecular weight unit is sec-1- TO rate doesn’t change SA=vmax/amount of protein unit = umol/secmg- Catalytic efficiency- kcat/kmo Physiological conditions [S]<kmo Therefore if [S]<<km then v=Vmax[S]/km o Vmax=kcat[E] therefore v=(kcat/km)[E][S] This tells how enzyme performs when S is low and tells how efficient enzyme is at low [S] Upper limit to kcat/km is diffusion limit In water rate constant for diffusion controlled rxn is approx. 10^9/Msec Km=M kcat=sec-1- Enzyme Kinetics- Modulating Enzyme activityo Ph, temp and inhibitors affect activityThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Enzyme inhibitors can give info on enzyme active site shape and surrounding residues, info on chemical mechanisms, on control and regulation of metabolic regulation, and provides opportunity for drug design There are reversible (Dead end or product type) and irreversible rxns Inhibition patterns- competitively binding or binds at alternative site effecting activity w/o effecting substrate binding or both Most common types: competitive, mixed and non-competitive, or uncompetitive(least common) Competitive inhibitor- competes with substrate for binding site- In michaelis menton scheme disrupts bolded E+S->ES->P+E Uncompetitive- binds to enzyme substrate complex but not free enzyme- E+S->ES->P+E Mixed inhibitors- bind to enzyme substrate complex- The smaller the value of Ki the tighter the binding How do we distinguish between types? - inhibition plot- Analysis of slope, y intcep, x intecep in presence or absence of inhibitor Ki= equilibrium constant for inhibitor binding to enzyme ki=kd Competitive inhibition ex- malonate competitive inhibition of succinate for succinate dehydrogenase—dead end- V=k2[ES] reduce amount of E therefore increase km- Competitive inhibition can be overcome w high concentration [S] vmax not effected- As add more inhibitor increase slope and reaction slows- Can only calc km and vmax without inhibitor; with inhibitor is called “apparent” Uncompetitive- decreased vmax and decreased km; decreased ES and decreasedvelocity- If [S] increases it will not overcome
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