Chapter 14. Mechanisms of Enzyme Action Biochemistry by Reginald Garrett and Charles Grisham. Essential Question. Although the catalytic properties of enzymes may seem almost magical, it is simply chemistry– the breaking and making of bonds – that give enzymes their prowess
Mechanisms of Enzyme Action
Reginald Garrett and Charles Grisham
Although the catalytic properties of enzymes may seem almost magical, it is simply chemistry– the breaking and making of bonds– that give enzymes their prowess
14.1 – What Are the Magnitudes of Enzyme-Induced Rated Accelerations?
H-O-H + Cl- H-O H Cl HO- + HCl
Reactant Transition state Products
14.2 – What Role Does Transition-State Stabilization Play in Enzyme Catalysis?
Figure 14.1Enzymes catalyze reactions by lowering the activation energy. Here the free energy of activation for (a) the uncatalyzed reaction, DGu‡, is larger than that for (b) the enzyme-catalyzed reaction, DGe‡.
The catalytic role of an enzyme is to reduce the energy barrier between substrate S and transition state
Figure 14.2The intrinsic binding energy of the enzyme-substrate (ES) complex (DGb ) is compensated to some extent by entropy loss due to the binding of E and S (TDS) and by destabilization of ES (DGd) by strain, distortion, desolvation , and similar effects. If DGb were not compensated by TDS and DGd, the formation of ES would follow the dashed line.
Figure 14.3 (a) Catalysis does not occur if the ES complex and the transition state for the reaction are stabilized to equal extents. (b) Catalysis will occur if the transition state is stabilized to a greater extent than the ES complex (right). Entropy loss and destabilization of the ES complex DGd ensure that this will be the case.
Raising the energy of ES raises the rate
Formation of the ES complex results in a loss of entropy. Prior to binding, E and S are free to undergo translational and rotational motion. By comparison, the ES complex is a more highly ordered, low-entropy complex.
Substrates typically lose waters of hydration in the formation of the ES complex. Desolvation raises the energy of the ES complex, making it more reactive.
Figure 14.4(c) Electrostatic destabilization of a substrate may arise from juxtaposition of like charges in the active site. If such charge repulsion is relieved in the course of the reaction, electrostatic destabilization can result in a rate increase.
Figure 14.5The proline racemase reaction. Pyrrole-2-carboxylate and D -1-pyrroline-2-carboxylate mimic the planar transition state of the reaction.
Figure 14.6(a) Phosphoglycolohydroxamate is an analog of the enediolate transition state of the yeast aldolase reaction. (b) Purine riboside, a potent inhibitor of the calf intestinal adenosine deaminase reaction, binds to adenosine deaminase as the 1,6-hydrate. The hydrated form of purine riboside is an analog of the proposed transition state for the reaction.
Figure 14.7 NACs are characterized as having reacting atoms within 3.2 Å and an approach angle of ±15° of the bonding angle in the transition state.
Figure 14.8 The active site of liver alcohol dehydrogenase (ADH) – a near-attack complex.
NAD+ + CH3CH2OH NADH + H+ + CH3CHO
BX + Y BY + X
BX + Enz E:B + X + Y Enz + BY
Figure 14.11 Examples of covalent bond formation between enzyme and substrate. In each case, a nucleophilic center (X:) on an enzyme attacks an electrophilic center on a substrate.
Figure 14.12 Catalysis of p-nitrophenylacetate hydrolysis can occur either by specific acid hydrolysis or by general base catalysis.
M2++ NucH M2+(NucH) M2+(NucH) + H+
Figure 14.14 Thermolysin is an endoprotease with a catalytic Zn2+ ion in the active site. The Zn2+ ion stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon.
0.25 nm (LBHB)
0.1 nm 0.18nm
1 order 0.07
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA
Figure 14.16 Structure of chymotrypsin (white) in a complex with eglin C (blue ribbon structure), a target protein. The residues of the catalytic triad (His57, Asp102, and Ser195) are highlighted. His57 (blue) is flanked above by Asp102 (red) and on the right by Ser195 (yellow). The catalytic site is filled by a peptide segment of eglin. Note how close Ser195 is to the peptide that would be cleaved in a chymotrypsin reaction.
Figure 14.18 The substrate-binding pockets of trypsin, chymotrypsin, and elastase.
A mixture of covalent and general acid-base catalysis
Figure 14.20 Burst kinetics in the chymotrypsin reaction.
Figure 14.23A detailed mechanism for the chymotrypsin reaction. Note the low-barrier hydrogen bond (LBHB) in (c) and (g).
Tetrahedral oxyanion transition state
Tetrahedral oxyanion transition state
Binding of substrate.
The formation of the covalent ES complex involves general base catalysis by His57
His57 stabilized by a LBHB.
The “oxyanion hole”: amide hyrdogens of Ser195 & Gly193
Collapse of the tetrahedral intermediate releases the first product.
The amino product departs, making room for an entering water molecule.
Nucleophilic attack by water is facilitated by His57, acting as a general base.
Collapse of the tetrahedral intermediate cleaves the covalent intermediate, releasing the second product.
Carboxyl product release completes the serine protease mechanism.
At the completion of the reaction, the side chains of the catalytic triad are restored to their original states.
The chymotrypsin mechanism involves two tetrahedral oxyanion intermediates
These intermediates are stabilized by a pair of amide groups that is termed the “oxyanion hole”
The amide N-H groups of Ser195 and Gly193 provide primary stabilization of the tetrahedral oxyanion
Figure 14.24 Mechanism for the aspartic proteases. LBHBs play a role in states E, ES, ET’, EQ’, and EP’Q.
A novel aspartic protease
Figure 14.26HIV mRNA provides the genetic information for synthesis of a polyprotein . Proteolytic cleavage of this polyprotein by HIV protease produces the individual proteins required for viral growth and cellular infection.
Protease inhibitors as AIDS drugs
HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck.
Direct comparison of enzyme-catalyzed reactions and their uncatalyzed counterparts is difficult
Chorismate mutase has become a model for making this comparison, thanks to the efforts of a large number of enzyme mechanism researchers
Chorismate mutase acts in the biosynthesis of phenylalanine and tyrosine in microorganisms and plants
It involves a single substrate and catalyzes a concerted intramolecular rearrangement of chorismate to prephenate
One C-O bond is broken and one C-C bond is formed
Figure 14.29 The critical H atoms are distinguished in this figure by blue and green colors.
Jeremy Knowles has shown that both the chorismate mutase and its uncatalyzed solution counterpart proceed via a chair mechanism. A transition state analog of this state has been characterized.
Figure 24.30 (a) the chorismate mutase homodimer (b)The active site, showing the bound transition-state analog.
Twelve electrostatic and hydrogen-bonding interactions stabilize the transition-state analog.
Figure 14.32 The carboxyvinyl group folds up and over the chorismate ring and the reaction proceeds via an internal rearrangement.
Figure 14.33 Chorismate boudn to the active site of chorismate mutase in a structure that resembles a near-attack complex. Arrows indicate hydrophobic interactions and red dotted lines indicate electrostatic interactions.
Figure 14.34 Chorismate mutase facilitates NAC formation. The energy required to move from the NAC to the transition state is essentially equivalent in the catalyzed and uncatalyzed reactions.