BICH 410 1st Edition Lecture 20 Outline of Last Lecture I Enzymes Current Lecture Vmax value of vmax varies on conc of enzyme Specific activity vmax g amount of protein o Number of substrate molec converted to product amount of tot protein present when E is saturated with substrate o Specific activity SA increases until total protein present is E pure protein at which time it will reach max o 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 soln o 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 km o Physiological conditions S km o 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 activity o Ph temp and inhibitors affect activity These 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 decreased velocity If S increases it will not overcome inhibition
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