Sept 10 - Biochem 423 Robert A. Orlando, Ph.D. ENZYMES –...

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Unformatted text preview: Biochem 423 Robert A. Orlando, Ph.D. ENZYMES – KINETICS EXAMPLE FROM MARKS’ Metabolic pathways will proceed to their ends if exergonic A → B → C → D → E Need to regulate to control metabolic events. Cars = pathway intermediates Barrier slows pathway = to rate limiting enzyme Some take alternate exit (branch points in pathway). TWO MAJOR WAYS TO REGULATE ENZYME ACTIVITY: PHYSICAL INHIBITOR – MORE ON THIS LATER LIMIT SUBSTRATE 49 | P a g e Biochem 423 Robert A. Orlando, Ph.D. EFFECTS OF [SUBSTRATE] ON ENZYME ACTIVITY Product formation X X INTERPRETATION OF THIS GRAPH: k1 k2 E + S → ES → E + P ← ← k−1 k−2 k1 >>>> k2 SO “BARRIER” IS AT k2 AND [ES] BACKS UP IF NO “BARRIER” WAS PRESENT AT k2 THEN GRAPH WOULD BE LINEAR 50 | P a g e Biochem 423 Robert A. Orlando, Ph.D. THEREFORE VO = k2[ES] (RATE OF ES → P) AND VMAX OCCURS WHEN ENZYME IS SATURATED ALGEBRAIC EQUATION DESCRIBING THIS GRAPH: MICHAELIS‐MENTEN VO = VMAX [S] Km + [S] ....SO WHAT IS Km (MICHAELIS CONSTANT) Km = ½ VMAX PERMITS COMPARISON OF ACTIVITIES BETWEEN ENZYMES HIGH Km = LOW BINDING AFFINITY LOW Km = HIGH BINDING AFFINITY 51 | P a g e Biochem 423 Robert A. Orlando, Ph.D. BECAUSE CURVE IS HYPERBOLIC, CANNOT ACCURATELY DEFINE VMAX OR KM…. ALGEBRAIC TRANSFORMATION OF MICHAELIS‐MENTEN EQUATION: DOUBLE‐RECIPROCAL PLOT (LINEWEAVER‐BURK PLOT) VO = VMAX [S] Km + [S] → 1 = Km + [S] VO VMAX [S] SEPARATE NUMERATORS 1 = Km + [S] VO VMAX [S] → 1 = Km + [S] . VO VMAX [S] VMAX [S] SIMPLIFY (REMOVE [S]) 1 = Km + 1 VO VMAX [S] VMAX WHERE → FORM y = mx + b y = 1 x = 1 b (y‐intercept) = 1 . VO [S] VMAX 52 | P a g e Biochem 423 Robert A. Orlando, Ph.D. LINEWEAVER‐BURK PLOT WHEN y = 0, solve for x 53 | P a g e ...
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