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What Are the Mechanisms of EnzymeInduced Rated Accelerations?

• Mechanisms of catalysis:

– Entropy loss in ES formation – Destabilization of ES – Covalent catalysis – General acid/base catalysis – Metal ion catalysis – Proximity and orientation

Why Is the Binding Energy of ES Crucial to Cataylsis?
Competing effects determine the position of ES on the energy scale Try to mentally decompose the binding effects at the active site into favorable and unfavorable The binding of S to E must be favorable But not too favorable! Km cannot be "too tight" - goal is to make the energy barrier between ES and EX‡ small

• • •


What Roles Do Entropy Loss and Destabilization of the ES Complex Play? Raising the energy of ES raises the rate • For a given energy of EX‡, raising the energy of ES will increase the catalyzed rate • This is accomplished by – a) loss of entropy due to formation of ES – b) destabilization of ES by • strain • distortion • desolvation

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.

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.


What Are the Mechanisms of Catalysis?
Serine Proteases are good examples of Covalent catalysis • Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway • The formation of the covalent bond provides chemistry that speeds the reaction

General Acid-base Catalysis
Catalysis in which a proton is transferred in the transition state • "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center • "General" acid-base catalysis involves acids and bases other than H+ and OH• These other acids and bases facilitate transfer of H+ in the transition state


Catalysis of p-nitrophenylacetate hydrolysis by imidazole—an example of general base catalysis. Proton transfer to imidazole in the transition state facilitates hydroxyl attack on the substrate carbonyl carbon.

Liver alcohol dehydrogenase catalyzes the transfer of a hydride ion ( H:-) from NADH to acetaldehyde (CH3CHO), forming ethanol (CH3CH2OH). An active-site zinc ion stabilizes negative charge development on the oxygen atom of acetaldehyde, leading to an induced partial positive charge on the carbonyl C atom. Transfer of the negatively charged hydride ion to this carbon forms ethanol.


An example of proximity effects in catalysis. (a) The imidazole-catalyzed hydrolysis of p-nitrophenylacetate is slow, but (b) the corresponding intramolecular reaction is 24-fold faster (assuming [imidazole] = 1 M in [a]).

What Can Be Learned from Typical Enzyme Mechanisms?

• Serine proteases and aspartic proteases are good examples • Knowledge of the tertiary structure of an enzyme is important • Enzymes are the catalytic machines that sustain life


The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA • All involve a serine in catalysis - thus the name • Ser is part of a "catalytic triad" of Ser, His, Asp • Serine proteases are homologous, but locations of the three crucial residues differ somewhat • Enzymologists agree, however, to number them always as His-57, Asp-102, Ser-195

Comparison of the amino acid sequences of chymotrypsinogen, trypsinogen, and elastase. Each circle represents one amino acid. Numbering is based on the sequence of chymotrypsinogen. Filled circles indicate residues that are identical in all three proteins. Disulfide bonds are indicated in yellow. The positions of the three catalytically important active-site residues (His57, Asp102, and Ser195) are indicated.


Serine Protease Mechanism
A mixture of covalent and general acid-base catalysis • Asp-102 functions only to orient His-57 • His-57 acts as a general acid and base • Ser-195 forms a covalent bond with peptide to be cleaved • Covalent bond formation turns a trigonal C into a tetrahedral C • The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195

The catalytic triad of chymotrypsin .


The substrate-binding pockets of trypsin, chymotrypsin, and elastase. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical
Institute. Not to be reproduced without permission. )

Diisopropylfluorophosphate (DIFP) reacts with active-site serine residues of serine proteases (and esterases), causing permanent inactivation.


A detailed mechanism for the chymotrypsin reaction. Note the low-barrier hydrogen bond (LBHB) in (c) and (g).






The “ oxyanion hole” of chymotrypsin stabilizes the tetrahedral oxyanion transition states of the mechanism

What Factors Influence Enzymatic Activity?
Six points: Rate slows as product accumulates Rate depends on substrate availability Genetic controls - induction and repression Enzymes can be modified covalently Allosteric effectors may be important Zymogens, isozymes and modulator proteins may play a role

• • • • • •


Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes (protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible enzyme represents an -OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group.

Proinsulin is an 86residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges.


The proteolytic activation of chymotrypsinogen.

The cascade of activation steps leading to blood clotting. The intrinsic and extrinsic pathways converge at Factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrinogen into fibrin, which aggregates into ordered filamentous arrays that become cross-linked to form the clot.


The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis. It needs LDH to regenerate NAD+ from NADH so glycolysis can continue. The lactate produced is released into the blood. The muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by excess pyruvate so the fuel won’t be wasted.

What Are the General Features of Allosteric Regulation?
Action at "another site" Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors These effectors are usually produced elsewhere in the pathway Effectors may be feed-forward activators or feedback inhibitors Kinetics are sigmoid ("S-shaped")

• • • •


Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot characteristic of normal Michaelis - Menten-type enzyme kinetics.

Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2 tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8 M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are enzymatically active as monomers.


Glycogen Phosphorylase
Allosteric Regulation and Covalent Modification
• Pi is a positive homotropic effector • ATP is a feedback inhibitor, and a negative heterotropic effector • Glucose-6-P is a negative heterotropic effector (i.e., an inhibitor) • AMP is a positive heterotropic effector (i.e., an activator)

The mechanism of covalent modification and allosteric regulation of glycogen phosphorylase. The T states are blue and the R states blue-green.


In this diagram of the glycogen phosphorylase dimer, the phosphorylation site (Ser14) and the allosteric (AMP) site face the viewer. Access to the catalytic site is from the opposite side of the protein. The diagram shows the major conformational change that occurs in the Nterminal residues upon phosphorylation of Ser14. The solid black line shows the conformation of residues 10 to 23 in the b, or unphosphorylated, form of glycogen phosphorylase. The conformational change in the location of residues 10 to 23 upon phosphorylation of Ser14 to give the a (phosphorylated) form of glycogen phosphorylase is shown in yellow. Note that these residues move from intrasubunit contacts into intersubunit contacts at the subunit interface. [Sites on the two respective subunits are denoted, with those of the upper subunit designated by primes (‘).]
(Adapted from Johnson, L. N., and Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annual Review of Biophysics and Biomolecular Structure 22:199-232.)

Regulation of GP by Covalent Modification
• In 1956, Edwin Krebs and Edmond Fischer showed that a ‘converting enzyme’ could convert phosphorylase b to phosphorylase a • Three years later, Krebs and Fischer show that this conversion involves covalent phosphorylation • This phosphorylation is mediated by an enzyme cascade


The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase.