Dual nature of the enzyme active site

specific sub-site and reaction sub-site

SPECIFIC SITE: the enzyme uses all possible (available) weak interactions via polar, charged, and hydrophobic groups to bind substrate Enzyme-substrate binding is often so specific that even a small change in the chemical composition of the substrate will abolish binding. Dissociation constants range from ~10-3 M to 10-9 M, the lower the value the more tightly the substrate is bound. REACTION SITE: other groups on the enzyme carry out the chemistry In some cases same amino acid residues participate in both specific binding and catalysis.

This dual nature is being effectively exploited in drug design:

amino acid changes can be made in the specific site without effecting its catalytic site
A hypothetical ketose isomerase enzyme

Amino Acids in General Acid-Base Catalysis

From Lehninger Principles of Biochemistry

The Michaelis-Menten Equation

Leonor Michaelis (1875-1949) and Maude Menten's (1879-1960) pioneers in enzyme kinetics

It assumes the formation of an enzyme-substrate complex ES (known as Michaelis complex), is in rapid equilibrium with free enzyme Breakdown of ES to form products is assumed to be slower than (1) formation of ES and (2) breakdown of ES to re-form E and S Which means that [ES] remains constant until the substrate is nearly exhausted: Steady state assumption (d[ES]/dt =0)

The dual nature of the Michaelis-Menten equation

Combination of zero-order and first-order kinetics

Vo =

Vmax[S]
_________

Km + [S]
m

When [S] is low, the equation for rate is first order in [S] K >>[S] V = (V / K ). [S] ([S]1 first order)
m o max

When [S] is high, the equation for rate is zero-order with respect to S (the rxn rate becomes independent of the substrate concentration) Km <<[S] Vo ~ Vmax = ([S]0=1 zero order) The Michaelis-Menten equation describes a rectangular hyperbolic dependence of Vo on [S]

Km =

(k2 + k-1 )
___________

k1

k1 is M-1sec-1 units (first order rxn) k2 & k-1 are in sec-1units (zero order) accordingly Km is in M units

Mechanisms for revesible are reversible Inhibition Three real, one hypothetical (very rare): all four
Competitive: Inhibitores (I) compete or prevent substrate from binding to the active site
I binds to the active site of E not ES (I is often structurally similar to S) higher Km same Vmax lines intersect on y-axis, shift in horizontal intercept Effect of the inhibitor can be overcome (reversed) by increasing substrate concentration [S]

Pure Non-Competitive: First proposed as a hypothetical case (because it made a useful

contrast to competitive inhibition) and observed very rare for a real enzyme. I binds to both E and ES at a site other than the active site (I need not to be similar to S) same Km, intercept on x-axis, because the k values (the rate to form EI or ESI) for binding to E & ES are the same). Lower Vmax (vertical intercept is shifted) the rate of EI formation equal to that of ESI formation Increasing [S] can sometimes partially (but not completely) reverse inhibition

Mixed Non-Competitive:
I binds to both E and ES at a site other than the active site: changes both Km & Vmax (because the k value for binding to E & ES are not the same) lines intersect to the left of y-axis either above or below vertical x-axis the rate of EI formation doesnt equal to that of ESI formation

Uncompetitive:
I binds to ES not E: both Km & Vmax are lower, although their ratios (the slopes Km/Vmax) are the same. Typical for rxns with more than one substrate. Lower Km means increase in affinity of an enzyme to its substrate. How? If an I removes the ES by binding to it, then the equilibrium will shift to compensate for the change in [ES] (remember LeChateliers principle). So the enzyme binds more S than expected. However adding more [S] cant overcome this (yes more ES would form, but more ESI would also form.)

(a) Competitive Km < KmI VmaxI = Vmax (b) Pure Non-Competitive Km = KmI VmaxI < Vmax (c, d) Mixed Non-Competitive KmI <Km < KmI VmaxI < Vmax (e) Uncompetitive Km /Vmax =const (slope)

Complex I: NADH to Coenzyme Q_ NADH:CoQ oxidoreductase Complex II: Succinate to Coenzyme Q_Succinate:CoQ oxidoreductase Complex III:Coenzyme Q to Cytochrome c_CoQ:Cyt c oxidoreductase Complex IV: Cytochrome c to O2_Cyt c:O2 oxidoreductase

SDH is on the matrix side of the IMS

From Lehninger Principles of Biochemistry