(P&S Ch 5; Fer Ch 2, 9; Palm Ch 10,11; Zub Ch 9) A. Reaction Mechanisms and Catalysis (1) proximity effect (2) acid-base catalysts (3) electrostatic (4) functional groups (5) structural flexibility B. Active Site Investigations (1) kinetic studies (2) detection of intermediates (3) x-ray crystallographic studies (4) chemical modification of amino acid side chains (5) site-directed mutagenesis studies C. Specific enzymes (1) lactate dehydrogenase and alcohol dehydrogenase (2) ribonuclease A (3) triose phosphate isomerase (4) amino acyl trna synthetases (5) carbonic anhydrase 77
(1) Proximity effect--enzymes bring reacting species close together -an enzyme can accelerate a rxn between two species simply by holding the two reactants close together in an appropriate orientation -intramolecular rxns between groups that are tied together in a single molecule are faster than the corresponding intermolecular rxns between two independent molecule, eg., cyclization of succinic acid to form succinyl anhydride c.f. formation of acetic anhydride from two molecules of acetic acid -difference in rates are 3-4 orders of magnitude (intramolecular> intermolecular) -effect is due mostly to the differences between the entropy changes that accompany the inter- and intramolecular rxns -formation of P involves a much larger loss of translational and rotational entropy in the intermolecular rxn 78
-a negative change in entropy increases both the overall free energy change in the rxn (ΔG = ΔH -TΔS) and the activation free energy (ΔG = ΔH - TΔS ) for the formation of the transition state -in the intramolecular rxn much of this entropy decrease has already occurred during the preparation of the reagent -enzymes that take catalyze intermolecular rxns take advantage of the proximity effect by binding the reactants close together in the active site, so that the reactive groups are oriented appropriately for the rxn -once the substrates are fixed this way the rxn behaves kinetically like an intramolecular process -the entropy decrease associated with the formation of the transition state has been moved to an earlier step, the binding of the substrates to form the E-S complex (2) Acid-Base Catalysis -task of a catalyst is to make a potentially reactive group more reactive by increasing its intrinsic electrophilic or nucleophilic 79
character -simplest way is to add or remove a proton eg., hydrolysis of an ester -hydrolysis of an ester in neutral aqueous solution can occur if the O atom of H 2 O, acting as a nucleophile, attacks the positively charged carbon -initial product is an intermediate where the carbon atom has four substituents in a tetrahedral arrangement -rxn is completed by the rapid breakdown of the tetrahedral intermediate to release the alcohol -H 2 O is intrinsically a comparatively weak nucleophile, and its rxn with esters in the absence of a catalyst is very slow -hydrolysis of esters occurs much more rapidly at high ph, when the negatively charged OH - replaces H 2 O as the reactive nucleophile -the nucleophilic character of H 2 O itself can be increased by 80
interaction with a basic group other than OH - where the base offers a pair of electrons to one of the protons of the H 2 O and increases the electron density of the oxygen -general base--used to describe any substance that is capable of binding a proton in aqueous soln -enzymes use a number of functional groups to fill this role (necessary since the [OH - ] at physiological ph limits its availability] -only requirement is that the base start out in the unprotonated form, which means that the ambient ph must be above the pk a of the conjugate acid -candidates include: basic groups from the ionizable or polar amino acid side chains, an amino-terminal NH 2 group, a carboxyl-terminal carboxylate ion, or from the oxygen or nitrogen atom of a peptide bond -the pk a of these groups can vary over a considerable range environment 81
-using a general base rather than OH - has the advantage that the basic group that is provided by the protein can be positioned precisely with respect to the substrate in the active site, allowing the proximity effect to come into play -hydrolysis of an ester can also be catalyzed by an acid where the acid donates a proton to the oxygen of the ester's carbonyl group, increasing the positive charge on the carbon and increasing the susceptibility of the ester to attack by a nucleophile -general acid--any substance that is capable of releasing a proton, and enzymes almost always use such proton donors in preference to H + or OH - because a general acid can operate at moderate ph and is easy to fix in position -requirement is that the ph be below the pk a (3) Electrostatic interactions --enzymes act by stabilizing the distribution of electrical charge in transition states -suppose the active site of an enzyme included a positively charged amino acid side chain, such as Lys or Arg, located near the oxygen atom of a carbonyl group. A fixed positive charge 82
in this region would favor the formation of the tetrahedral intermediate, even if there were no transfer of a proton from the charged species to the oxygen. A fixed negative charge in the region of the nucleophile would have a similar effect. The interactions of such fixed charges are termed electrostatic effects. -as a reacting substrate is transformed into a transition state, the changing charges on its atoms interact with the charges on all of the other atoms in the surrounding protein, and also with the charges on any nearby water molecules -the energy difference between the initial state and the transition state thus depends critically on the details of the protein structure -modern computational techniques, when taken with the wealth of structural information from X-ray crystallography and other biophysical studies, have made it possible to calculate the contributions that various components of an enzyme's active site make to the activation free energy (ΔG ), and to predict quantitatively how ΔG might be altered by modifications of the protein 83
-can be tested experimentally by modifying the gene that encodes the protein (site-directed mutagenesis) (4) Nucleophilic/Electrophilic Catalysis (a) Nucleophilic Catalysis--strategy is to use stronger nucleophilic groups (than water) as part of the enzyme's active site -HOCH 2 group of a serine residue often used as a nucleophile -instead of immediately yielding the free COOH, the breakdown of the tetrahedral intermediate yields an intermediate ester that is covalently attached to the enzyme -the acyl-enzyme intermediate must be hydrolyzed by a second rxn in which H 2 O is the nucelophile -proteolytic enzymes (serine proteases) work in this way -two step pathway requires that the intermediate be more susceptible to nucleophilic attack by H 2 O than by the original ester or amide 84
-nucleophilic groups on enzymes participate in a variety of other types of rxn in addition to hydrolytic rxns, eg., acetoacetic acid decarboxylase -rxn proceeds by the formation of a Schiff base intermediate, in which the substrate is covalently attached to the ε-amino group of a lysine residue at the enzyme's active site -this intermediate is formed by a nucleophilic attack of the amino group on the carbonyl carbon, followed by the splitting out of H 2 O -protonation of the nitrogen atom of the Schiff base introduces a positive charge that pulls electrons from the nearby carboncarbon bond, causing decarboxylation -this is an extreme example of an electrostatic effect where the enzyme introduces a charged group into the substrate itself -basic feature of this mechanism is the formation of an intermediate state in which the substrate is covalently attached 85
to a nucleophilic group on the enzyme -the -CH 2 SH of cysteine is often used as a nucleophile also -the COOH of Asp and Glu participate in rxns involving the hydrolysis of ATP and -the imidazole ring of His can play a similar role -some enzymes use coenzymes: thiamine, biotin, pyridoxamine, or tetrahydrofolate as additional nucleophilic reagents (b) Electrophilic Catalysis--numerous enzymes use bound metal ions to form complexes with substrates -metal ion functions as an electrophilic group -eg., carbonic anhydrase--contains a Zn 2+ ion in the active site and forms a complex with the carbonyl oxygen atom of the aldehyde or peptide substrate -withdrawal of electrons by the Zn 2+ increases the partial positive charge on the carbonyl carbon atom and promotes the rxn of carbon with a nucleophile 86
(5) Structural Flexibility--some enzymes undergo major structural rearrangements when they bind substrates or inhibitors -eg., hexokinase ATP + glucose ADP + glucose-6-phosphate -hexokinase binds glucose, it undergoes a structural organization that brings together the elements of the active site -the enzyme literally closes like a set of jaws around the substrate referred to as an induced fit -carboxypeptidase A undergoes a major structural change when it binds its substrate -rearrangement of the protein pulls the hydrophobic part of the substrate out of the aqueous soln by surrounding it with nonpolar portions of the protein -advantages: (1) maximizes the favorable entropy change associated with removing a hydrophobic molecule from H 2 O (2) allows enzyme to intensify the electrostatic effects that promote the formation of the transition state (the substrate is 87
forced to respond to the directed electrostatic fields from the enzyme's functional groups, instead of the disordered fields from the solvent -structural change can help to explain the high specificity of some enzymatic rxns; eg., hexokinase undergoes a structural change upon binding glucose which promotes the binding of the other substrate (ATP) -ATP doesn't bind unless glucose is already present in the catalytic site -if ATP were to bind in the absence of glucose, the enzyme might have a tendency to catalyze the transfer of phosphate from ATP to water, resulting in a wasteful loss of ATP: ATP + H 2 O ADP + Pi -X-ray crystal structure of an enzyme give only static snapshots of the molecules, which can be very flexible -vibrations and rotations involving only a few atoms 10-13 to 10-11 s 88
-larger motions, i.e., flipping of aromatic rings 10-9 to 10-8 s -major reorganizations 10-6 to 10-3 s -all these can be important for catalysis 89