Fundamentals of Biochemistry Third Edition Donald Voet • Judith G. Voet • Charlotte W. Pratt Chapter 11 Enzymatic Catalysis Copyright © 2008 by John Wiley & Sons, Inc.

Enzymes are proteins which catalyze biologically interesting reactions They enhance reaction rates tremendously!

Enzymes are classified into six broad categories Check out http://www.chem.qmul.ac.uk/iubmb/enzyme/ for a complete listing of all known enzymes and the rules on classification

The traditional view of an enzyme’s catalytic mechanism is called the “lock and key” model (Emil Fischer, 1894), in which the enzyme contains an active site which binds the substrate molecule, upon which the reaction will occur. The specificity of an enzyme for a particular substrate can therefore be determined by the enzyme’s active site structure.

Enzymes are stereospecific — aconitase catalyzes the interconversion of citrate and isocitrate Citrate is a prochiral molecule (needs a group changed to be chiral), so is not itself chiral. Aconitase is still stereospecific for a particular orientation of citrate by binding to citrate at three points

Geometric specificity allows enzymes to distinguish different groups on substrate molecules — and to allow a broader array of substrate molecules

Some enzymes require cofactors — other chemicals that allow the enzyme’s catalytic function

Some enzymes require cofactors — other chemicals that allow the enzyme’s catalytic function Prosthetic groups, like biotin, are permanently associated with the enzyme, in this case, carboxylases

Some enzymes require cofactors — other chemicals that allow the enzyme’s catalytic function Cosubstrates are discarded, once the reaction is over

NAD+ acting as a cofactor for alcohol dehydrogenase (ADH)

Kinetics of enzyme-catalyzed reactions Svante Arrhenius came up with the idea of a transition state in 1889; Rene Marcelin invented the ‡ notation in 1910 Transition state theory posits the existence of a high-energy transition state from which the reaction can literally go forwards (products) or backwards (reactants)

Kinetics and thermodynamics of enzyme-catalyzed reactions transition state activation energy reactants free energy change endergonic = positive ΔGrxn exergonic = negative ΔGrxn products

Kinetics and thermodynamics of enzyme-catalyzed reactions transition state kinetics reactants thermodynamics products

Kinetics of enzyme-catalyzed reactions An intermediate is a metastable chemical species that occurs during a reaction. The role of an enzyme may be to lower one transition state’s energy to favor the reaction in a particular direction.

Kinetics of enzyme-catalyzed reactions An intermediate is a metastable chemical species that occurs during a reaction. The role of an enzyme may be to lower one transition state’s energy to favor the reaction in a particular direction. catalyzed rate determining step uncatalyzed rate determining step

Kinetics of enzyme-catalyzed reactions The efficacy of an enzyme is quantified by calculating how much it lowers the transition state energy: ΔΔGcat‡

Mechanisms of catalysis First, rules about curved arrows: • move pairs of electrons only • two curved arrows do not originate from the same bond or atom • carbon and nitrogen can make four bonds maximum • oxygen can make three bonds maximum

Mechanisms of catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation Preferential binding at the transition state

Mechanisms of catalysis recall general acid-base catalysis: no catalysis acid catalysis (A = acid) base catalysis (B = base)

Mechanisms of catalysis Enzymes have an optimal catalytic pH, implying that the molecule must have a certain overall charge, and certain side changes must have, or have not, a charge; fumarase’s pH dependence is shown.

Bovine pancreatic RNase S shows acid/base catalysis

Often, the text will use a large arc to represent the enzyme, with particular residues’ side chains drawn out to show particular interactions with the substrate.

In the case of this RNase, the RNA is the substrate and the phosphoester bond needs to be cut. Given the enzyme’s optimal pH of around 6, two histidine side chains seem to be key with His 12 acting as a base and His 119 acting as an acid.

Mechanisms of catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation Preferential binding at the transition state

Covalent catalysis requires a nucleophile because a catalyst-substrate covalent bond is made. Often, the intermediate is a Schiff base because the nucleophilic group is an amine from lysine or an imidazole from histidine.

This example is of a carboxylase decarboxylating acetoacetate.

Mechanisms of catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation Preferential binding at the transition state

By contrast, metal ions act as electrophiles By contrast, metal ions act as electrophiles. They bind to substrates through metal-carbon bonds or can stabilize electron-rich species or mediate redox reactions by transferring electrons.

Example: Zn2+ in carbonic anhydrase has a square planar geometry; three ligand bonds are to imidazoles on histidines and the other is to water or bicarbonate ion.

The catalytic mechanism

Mechanisms of catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation Preferential binding at the transition state

In the classic SN2 mechanism, the inversion of stereochemistry occurs due to the alignment of the ligand p orbitals with the planar substrate central carbon. The more the substrate can be oriented correctly by the enzyme to react with another substrate.

Mechanisms of catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation Preferential binding at the transition state

Enzymes that preferentially bind the transition state structure increase its concentration and therefore proportionally increase the reaction rate — Wolfenden in 1972 and Lienhard in 1973

Preferential binding of the transition state lowers the activation energy of the reaction, increasing the rate

Transition state analogs are enzyme inhibitors — an enzyme inhibitor slows the rate of the catalytic reaction by binding to the active site and preventing the actual substrate from binding Proline racemase catalyzes this reaction These molecules bind to the racemase with greater affinity than proline

Lysozyme is an enzyme that destroys bacterial cell walls by hydrolyzing a particular linkage in the glycosidic (sugar) chain in the cell wall material NAG = N-acetylglucosamine NAM = N-acetylmuramic acid

Lysozyme is a small 14.3 kD, 129-residue protein, and has an active site in a cleft within the protein that runs the length of the molecule; researchers had to use transition state analogs of the sugar chain for the X-ray crystallographic study Blake, Koenig, Mair, North, Phillips and Sarm, Structure of Hen Egg-White Lysozyme: A Three-dimensional Fourier Synthesis at 2 Å Resolution, Nature 206, 757 - 761 (22 May 1965)

The key to the cleavage of the sugar chain lies in the protein’s ability to strain a particular sugar’s ring structure from the chair to the half-chair confirmation

This steric strain is accomplished by two residues: Glu 35 and Asp 52 This steric strain is accomplished by two residues: Glu 35 and Asp 52. Specifically, because Asp 52 has a negative charge, it can stabilize an oxonium ion in the transition state. Further, Glu 35 is in a nonpolar pocket and thus can retain its acid proton at a relatively high pH, meaning it can be the acid catalyst

Isotopes are useful in elucidating mechanisms; by using oxygen-18 and mass spectrometry, it is possible to determine the fate of the oxygen from the water.

Lysozyme’s activity proceeds by forming a covalent bond with the sugar chain, and by stabilizing the oxonium transition state.

Serine proteases are another class of proteins that have a common mechanism. These proteins, including trypsin (shown), chymotrypsin and elastase, all have a particularly active serine residue

The test for serine proteases is to react it with DIPF, which irreversibly binds to it, because of the formation of a covalent bond with the hydroxyl group on the serine’s side chain.

This knowledge of how serine proteases bind to substrates is exploited in toxins: acetylcholinesterase is a serine protease and can be blocked by several molecules that bind to the serine side chain.

In addition to the serine residue, an Asp and a His residue play a major role in the catalysis, so much so that the three residues are called a “catalytic triad”. These residues, not surprisingly, are invariant over nearly all species.

Different serine proteases have the same shape “pocket” but specificity may be determined by the residues lining the pocket. Scissile bond = bond to be cleaved

Folding of the protein, too, determines the local structure of the pocket and therefore has implications for enzyme specificity.

A note about enzyme activation: since enzymes (especially proteases) should not be “on” all the time, but since enzymes take a long time (molecularly) to synthesize, it makes sense to have a store on inactive forms of the enzyme that can be activated by a simply cleavage, like trypsin. Its precursor, trypsinogen, is called a zymogen. Zymogens are inactive because their active sites are distorted.

Blood coagulation is the result of the protein fibrin, which comes from fibrinogen cleaved by the serine protease thrombin.