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 At high [S], all E is ES so... -[ES]=[Etotal] -plateu is due to satuaration effect Vmax -units: uM/min -observed when virtually all E is present as ES complex -max rate, extrapolate to at high [S] -hyperbola -record it per active site of E Kcat -turnover number -units: sec-1 -describes the limiting rate of an enzyme (catalytic rxn at satuaration) -concentration of S converted to P per unit time per enzyme site -at high [S], reaction is independent of [S] b/c all E sites are filled Reach at which [S]=km -km is the substrate concentration that provides one-half the maximal rate -it's [S] that provides 1/2 the rate -50% of the sites are occupied by substrate (1/2 the rate) Consider [S] <<<<< Km -v is dependent upon substrate concentration and enzyme concentration -v is dependent upon formation of ES -> P -initial region where if double [S], rate doubles -linear region Specificity constant Kcat/Km (uM-1sec-1) -use to compare enzymes -predicts how efficient enzyme is at binding S and going forward to P -at extrememy E, it predicts the probability of forming a product (ES -> P) Designing an experiment regarding enzyme kinetics -take into account assumptions of steady state Experiment 1: [E] is very low relative to [S] -goal: measure initial period of reaction where P formation is linear y=mx+b -slope provides the rate K or V or Vo -units: uM product produced per unit time (uM/s or uM x s-1) Experiment 2: function of [S] -increase [S] -family of lines y=mx+b -report as uM x s-1 or s-1 -remember, low [S] have dramatic increase of rate with additional [S] (below slope line see this) High [S] -Km has no role when all E sites are satuarated -slowest step (k+2) determines rate (rate limiting step) S=[Km] -Km is the [S] that provides 1/2 Kcat or Vmax -50% sites ES/50% Efree [S] <<<<<<< Km -rate is directly proportional to [S] -rate is linear y=mx+b -slope = Kcat/Km (uM-1s-1) -Kcat tells how fast can go when all sites are satuarated -Km predicts affinity of S for E kcat/km units: uM⁻1s⁻1 -predicts lower limit of K₊₁ -higher number means better substrate since it is lower limit predicotr -higher # = weaker binding K₊₁ -E colliding with S -concentration dependence units: uM-1 s-1 K₋₁ and K₊₂ units: s-1 -exponential functions -concentration has nothing to do w/ ES ->P or ES backwards 3 steady state parameters Kcat: s-1, turnover numerb Km: uM, SS dissociation constant Kcat/Km: uM-1s-1, specificity constant Are there enzymes that do not obey M-M kinetics? -yes, when have evidence of cooperativity or allosteric regulation -look for sigmoidal behavior -may not be able to detect sigmoidal behavior at the conditions of the expt. Linear Transformation of kinetic data -Lineweaver-Burk double reciprocal plot -Eadie-Hofstee plot(v vs. v/[S]) -Hanes-Woolf plot([S]/v vs. [S]) Lineweaver-Burk -used more often -using really only low [S] data Weaknesses of Lineweaver-Burk -data the most difficult to obtain techinically (low[S]) given most weight in determining linear fit -difficult to evaluate quality of data directly (scatter, satuaration) -cannot determine Kcat (Vmax) or Km directly from the plot