Enzyme kinetics-2

CH 432
 Influence of Temperature and pH on Enzyme
Kinetics
• Enzymes typically have optimum temperature and pH range
over which their activity is maximum:
• Enzymes primary structure consists of sequence of amino
acids, while secondary and tertiary structures arising due to S-
S and hydrogen bonds provide 3D structure of the proteins.
• Temperature effect: At low temperature low catalytic activity
is natural. At high temperature proteins denature leading to
loss of activity.
• pH effects: at too high or too low pH enzymes get denatured
and their activity is irreversibly affected.
• At pH slightly away from optimum range activity reduces but
the effect is usually reversible.
• The optimum pH range arises due to several ionisable groups
present in the protein. A simplistic model is shown, where the
highest activity is attributed to the zwitterionic form of the
enzyme:
 pH effect included in the
Michaelis Menten mechanism
𝑘2 𝐸 0 𝑆
• SSA gives 𝑣 =
𝐾a 𝐻+ 𝐾′𝑎 𝐻+
𝐾𝑚 1+ + + 𝑆 1+ +
𝐻+ 𝐾b 𝐻+ 𝐾′𝑏

• At low [S], first term in denominator dominates

𝑘2 𝐸 0 𝑆
• 𝑣 ≈ 𝐾 𝐻+
a
𝐾𝑚 1+ + +
𝐻 𝐾b

• Further at acidic pH, last term in denominator survives

𝑘 𝐸 𝑆𝐾
• 𝑣 ≈ 2 0 + b,
𝐾𝑚 𝐻
• Hence log10v = constant + pH gives a straight line with slope=+1 as shown
• at basic pH, 2nd term in denominator survives
𝑘 𝐸 𝑆 𝐻+
• 𝑣 ≈ 2 0 ,
𝐾𝑚 𝐾b
• Hence log10v = constant - pH gives a straight line with slope=-1 as shown.
• At intermediate pH, first term dominates
𝑘 𝐸 𝑆
• 𝑣 ≈ 2 0 ,
𝐾𝑚
• Hence log10v = constant gives a horizontal line as shown
 pH effect included in the
Michaelis Menten mechanism
• The point of intersection of these lines gives pKa
and pKb
• through conditions
𝑘2 𝐸 0 𝑆 𝐾b 𝑘2 𝐸 0 𝑆
• 𝑣≈ ≅
𝐾𝑚 𝐻 + 𝐾𝑚
𝑘2 𝐸 0 𝑆 𝐻 + 𝑘2 𝐸 0 𝑆
• and 𝑣≈ ≅
𝐾𝑚 𝐾b 𝐾𝑚
• This is an ideal scenario with only one pair of
ionisable groups. In practice proteins have many
such ionisable groups, and so the transitions are
not very sharp. One sees a bell type curve with a
range of pH where activity is optimum, and a sharp
drop in activity when the pH moves away from this
range.
 pH effect included in the Michaelis Menten mechanism

𝑘2 𝐸 0 𝑆
• Going back to the SSA equation: 𝑣 = 𝐻+ 𝐾′𝑎 𝐻+
𝐾a
𝐾𝑚 1+ + + 𝑆 1+ +
𝐻+ 𝐾b 𝐻+ 𝐾′𝑏

• Similarly one can work out at high [S] when 2nd term in denominator
𝑘2 𝐸 0
dominates to get 𝑣 ≈ 𝐾′ 𝐻+
𝑎 +
1+ +
𝐻 𝐾′𝑏

• Further at acidic, basic and intermediate pH one will get lines with slope
+1, -1, and 0, from which pK’a and pK’b could be extracted.
 Enzyme Inhibition (Competitive)
• E + S = ES  E + P (rate constants k1, k-1, k2)
• E + I = EI (rate constants k3, k-3)

𝑘2 [𝐸]𝑜 [𝑆]
• SSA treatment gives 𝑣 =
𝑆 +𝐾
[𝐼] 𝑘−1 +𝑘2 𝑘−3
where 𝐾 = 𝐾𝑚 1 + , 𝐾𝑚 = and 𝐾𝐼 =
𝐾𝐼 𝑘1 𝑘3

Shows that the rate is inhibited when [I] is non zero