Biochemistry I Fall Term, 2004Serine Proteases: pH Effects in Enzyme MechanismIn many enzymes, the ionization state of the amino acid side chains are important for catalyticactivity. In the case of trypsin, the His residue in the active site must be deprotonated in order forit to act as a general base catalyst.Consider the following reaction scheme:Ka is the acidity constant for the group on the enzyme (i.e. His in the case of trypsin)The effect of the protonation on the enzyme activity can be calculated using steady-statemethods.Note: The full derivation can found on pH Effects in Enzyme Mechanism, linked from theLecture 26 notes. The result we will consider is a modified Michaelis-Menten equation, in whichboth Vmax and KM are modified:and where:Thus, a hyperbolic substrate saturation plot will be obtained at all pH values!Compare the above to the analogous expressions we found for enzyme inhibition.How Vmax varies with [H+] can be investigated by looking at some values for [H+]: [H+] αααα V'max >>Ka (low pH) large --> 0 = Ka 2 Vmax/2 <<Ka (high pH) 1 VmaxThe above shows that when the pH is such that the velocity of the reaction is reduced by half, thepH is equal to the pKa of the group that is involved in catalysis.The above table is an accurate summary of the dependence of Vmax on pH for chymotrypsin inthe range where the protein is in its native conformation. (What additional complexity, related tosubstrate specificity, must be considered for trypsin-catalyzed reactions?)If more than one ionizable group is required for catalysis then bell shaped curves are observedwith the maximum enzyme activity halfway between the pKas of the ionizable
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