BIO515 EXAM 2 pt. 2 PDF

Enzyme Kinetics Kinetics is the study of the rate at which compounds react. - Rate of enzymatic reaction is affected: o Enzyme concentration o Substrate concentration o Effectors (pH, co-factors) o Temperature How to study enzymes 1. 2. 3. 4. Purify enzyme. Design assay that measures the activity of...

Enzyme Kinetics Kinetics is the study of the rate at which compounds react. - Rate of enzymatic reaction is affected: o Enzyme concentration o Substrate concentration o Effectors (pH, co-factors) o Temperature How to study enzymes 1. 2. 3. 4. Purify enzyme. Design assay that measures the activity of enzyme. Make mutations measure the effects on enzyme activity. Repeat (3.) under different conditions to determine kinetic parameters that give clues of how enzyme works and what AAs are essential. Why study enzyme kinetics? - - Precise, quantitative description of biocatalysis. Determine kinetic terms to compare enzyme efficiencies and substrate specificity. Understand catalysis mechanism. In combination with mutagenesis, uncover the function of the essential amino acids of the catalytic mechanism. o Differentiate which residues are involved in catalysis vs binding of substrate. Identity and characterize enzyme inhibitors. Understand regulation of enzyme activity Enzyme assay example - Alcohol dehydrogenase (ADH): converts ethanol to acetaldehyde. NADH has a higher absorbanve at 340 nm compared to NAD- Can monitor NADH production during reaction o I.e. sample containing [EtOH]. o Add alcohol Dehydrogenase (ADH) It will produce NADH which cane be monitored in UV spec How to do Kinetic Measurements Experiment: 1.) Mix enzyme + substrate in known concentrations. 2.) Record rate of produce formation (or substrate disappearance) as a function of time (the velocity of reaction, i.e. umol/mol) 3.) Change substrate concertation and repeat. 4.) Plot initial velocity (Vo) versus substrate concentration. Effect of Substrate Concentration E + S óES à E + P Vo, initial velocity. Measured at the beginning of reaction before much susbtrate is used up and when [S] > [E] At low [S], increasing [S] à increasing velocity (Vo). At high [S], increasing [S] has negligible effect on Vo. (enzyme becomes saturated) Enzymes that exhibit this relationship (hyperbolic curve) when you measure Vo as a function of [S] are called Michaelis-Menten Enzymes what’s an equation that explains this curve? Y is Vo Vmax is asymptote. X is [S] Km is [S] when Vo is ½ Vmax 𝑣! = "#$%['] )#*['] Enzymes that exhibit this hyperbolic curve are called Michaelis-Menten Enzymes Assumption 1: k2 is rate limiting (much slower) à and hence V0=k2[ES] (ususlaly, if rate limiting k2= kcat) Assumption 2: “steady state” conditions: [ES]formation = [ES]breakdown d[ES]/dT = 0 In steady state condition the amount of ES is not changing! Assumtion 3: Concentrations of S, E, ES and P early in the reaction (Vo) = - [S] is ~ constant. Why? [P] is negligible. [ES] is difficult to determine experimentally [Efree]= [ET]-[ES] These 3 sets of assumptions allowed MM to derive an equation to fit this curve. Main equations: 1) 𝑣! = 2) 𝑣! = +,$-[.-]['] )#*['] "#$%['] )#*['] 3) Vmax= kcat[ET] Kcat (turnover number): how many substrate molecules can one enzyme molecule convert per unit of time kcat = Vmax / [ET] Km (Michaelis constant): an approximate measure of substrates affinity for enzyme. Km = [S] at ½ Vmax Kcat is whatever is the rate-limiting step (i.e. slowest): k2 or k3, etc if only 1 step, then k2 is kcat Kcat is measure of the turnover number: how many substrate molecules the enzyme can turnover, or convert, to product in a given unit of time when E is saturated with substrate. (ie the fasted that they enzyme can work) Turnover numbers examples: - Lysozyme: 2/second - a really slow catalyst but gets the job done Catalase (breaks down toxic hydrogen peroxide): 40 million/second Meaning of Km Km ~ Kd of enzyme for substrate - The lower the Km the higher the affinity (usually) Km~the biological [S] in the cell. Why is Km often analogous to Kd? - Km is a measure of the balance between: [ES] breakdown (sum of K2 and K1 rates) and [ES] formation (k1). Saying the Km is ~ Kd releies on 2 assumptions: o Assumtiptim 1: K2