Enzyme Catalysis

Enzyme Catalysis According to transition state theory there is an energy barrier to be overcome to convert substrates to products, and the height of this barrier determines the rate of the reaction For an enzyme to catalyze a reaction this barrier must be lowered In 1948 Linus Pauling proposed that enzymes can accomplish this task if they can bind the transition state with higher affinity than the ground state (transition state stabilization) In principle a new catalyst can be designed if you can create a protein that will bind the transition state structure of a reaction of interest with very high affinity The problem is that transition states are unstable, transient species with very short lifetimes

Transition State Analogs If you can synthesize a stable analog of the transition state of a reaction of interest then you have a starting point to design a new enzyme catalyst ester hydrolysis reaction

Designing New Proteins Now the problem becomes to design a new protein that will recognize and bind this transition state analog with high affinity Nature has already solved the problem of producing new proteins that recognize new structures Antibodies are designed to recognize a wide range of foreign substances They are composed of two heavy and two light chains The amino terminal ends of each chain consist of highly variable regions called antigen binding sites These are the binding sites for foreign substances Foreign substances can elicit the production of as many as 100 million different antibody proteins

Antibody Screening How do we rapidly screen this large array of antibodies to identify those with high affinity to our designed transition state analog ? ELISA – enzyme linked immunosorbent assay Reaction of the chromogenic substrate is a measure of the binding of the antibody to the immobilized antigen

Transition State Analogs To produce a catalytic antibody you must introduce a substance that has a transition state analog structure, and then elicit antibodies to that structure As an example, esterases cleave ester bonds by going through a tetrahedral transition state After isolating and purifying the antibodies elicited to these phosphonates their catalytic properties can be tested with the corresponding substrates

Catalytic Antibody Properties Are these really catalysts ? • they accelerate the reaction when added in small amounts • the rate of reaction acceleration is proportional to the amount added • they produce many molecules of product for each antibody molecule • they show saturation kinetics these are the criteria that demonstrate a catalytic process

A model reaction stable transition state analog Antibodies have been elicited to this analog and then examined as catalysts for the model reaction

Catalytic Antibody active site The structure of an antibody that catalyzes the hydrolysis of this model reaction has been determined The first surprise was that the active site of this catalytic antibody resembles that of a serine protease There is an active site serine (S99) and an adjacent histidine (H35) There is also a lysine (K97) to stabilize the negative charge on the phosphonate group The only missing piece is the auxiliary catalyst to complete the catalytic triad So, how good a catalyst is this antibody compared with serine proteases ?

Catalytic Antibody relative activity The native serine proteases are very efficient catalysts Comparison of the catalytic antibody to mutants that are missing the Asp of the catalytic triad show that this protein is a respectable catalyst A double mutant of trypsin in which the position of the auxiliary catalyst has been altered is a better comparison to the structure of the catalytic antibody Science265, 1059 (1994)

Catalytic Antibody transition state binding How does a binding protein (antibody) become a catalyst (enzyme) ? hapten transition state The catalytic antibody makes numerous stabilizing interactions with the transition state analog J. Comp. Chem.23, 84 (2002)

Designing a New Enzyme Chorismate mutase catalyzes the rearrangement of chorismate to prephenate a stable analog of the transition state has been synthesized eliciting antibodies that bind to this analog with high affinity would stabilize this transition state and could lead to catalysis of the chorismatemutase reaction

ChorismateMutase Catalytic Antibody The transition state analog is bound in a well-defined orientation to the catalytic antibody There are a number of interactions that serve to orient the transition state analog in this binding pocket Science263, 646 (1994)

ChorismateMutase transition state stabilization catalytic antibody B. subtilischorismatemutase The native enzyme provides additional interactions with the transition state that are not present in the catalytic antibody This additional stabilization explains why the native enzyme is a more efficient catalyst than the antibody

Catalytic Antibody range of catalyzed reactions catalytic antibody references

Catalytic Antibody further refinements

Catalytic Antibody limitations • antibodies do not contain conveniently placed catalytic functional groups • the conformation dynamics that have evolved in enzymes is not present in antibodies • transition state analogs are NOT transition states

RNA catalysis ? • Can RNA act as a true catalyst? • If so, then how good can RNA be as a catalyst? • What range of reactions can RNA catalyze? • What structural features of an RNA molecule can confer catalytic properties?

Ribozymes RNA-protein comparison So, how can this simple set of RNA functional groups catalyze reactions ?

Ribonucleases enzyme vs. ribozyme Enzymes can use a variety of functional groups as acid-base catalysts Enzymes have also recruited a number of cofactors to extend their capabilities Ribozymes must rely primarily on metal ions and metal-bound waters Ann. Rev. Biochem.69, 597 (2000)

Ribozymes hammerhead reaction The hammerhead ribozyme catalyzes the selective cleavage of oligonucleotides The substrate (S) is bound on either side of the cleavage site by eight base pairs The bases in boxes are conserved throughout the ribozyme family Cleavage leads to a 5’-product (P1) with a cyclic phosphate group and a 3’-product (P2) with a terminal hydroxyl group Ann. Rev. Biochem.66, 19 (1997)

Ribozymes hammerhead mechanism G-12 acts as a general base to abstract a proton from the O2’ of C-17 (----) This hydroxyl of C-17 is the nucleophile for attack on the phosphate group (----) G-8 acts as a general acid to donate a proton to O5’ of the leaving group (····) Cell81, 991 (1995)

Ribozymes hammerhead mechanism stabilization of the transition state

Ribozymes general phosphodiesterase mechanism proton abstraction from 2’-hydroxyl nucleophilic attack assisted by metal-1 with the developing negative charge stabilized by metal-2 Proc. Natl. Acad. Sci.95, 542 (1998)

Ribozymes substrate binding base pairing recognition is the primary mechanism of substrate recognition and binding in ribozymes Ann. Rev. Biochem.66, 19 (1997)

Group I Introns reaction This class of ribozymes catalyze an intron splicing reaction during the synthesis of mRNA base pairing positions the substrate guanosine is the nucleophile after 3’ to 5’ coupling the products are released

Group I Introns active site The incoming guanosine substrate is positioned for attack at the phosphodiester bond Biochemistry41, 2516 (2002)

Group I Introns transition state Formation of the new bond to the guanosine nucleophile and displacement of the leaving nucleotide Metal ions play a role in generating the nucleophile for attack and also in stabilizing the phosphoryl group Biochemistry41, 2516 (2002)

Group II Introns structure Unlike with Group I introns, here the excision of the intron does not require GTP as both a substrate and a nucleophile Intron excision occurs through the formation of a lariat structure to bring the exon regions together

Ribonuclease P reaction Ribonuclease P catalyzes the cleavage of precursor transfer-RNAs to produce the mature final product This enzyme contains a small protein domain that was proposed to be the catalytic domain There is also a large RNA component that was thought to participate in substrate recognition Both of these ideas were completely WRONG !

Ribonuclease P substrate binding when the pre-tRNA leader sequence was modeled into the P protein the cleavage site is completely outside of the protein domain Proc. Natl. Acad. Sci.95, 15212 (1998)

Ribonuclease P overall structure Catalytic RNA domain Protein domain t-RNA substrate

Ribonuclease P RNA domain structure pre-tRNA substrate with its leader sequence (▪▪▪) substrate specificity (S) domain catalytic (C) domain site of RNase P protein binding Curr. Opin. Str. Biol.13, 325 (2003)

Ribonuclease P substrate cleavage site base pairing interactions between thetRNA substrateand thecatalytic RNA domain Curr. Opin. Str. Biol.13, 325 (2003)

Ribonuclease P catalytic transition state multiple metal ions are involved in the generation of the nucleophile, the stabilization of the transition state, and the enhancement of the leaving group Curr. Opin. Chem. Biol.4, 553 (2000)

RNA Catalysis ?

Abzymes and Ribozymes • stabilization of the transition state of a reaction will lead to catalysis • Abzymes are catalytic antibodies produced to bind transition state analogs of a reaction of interest • Abzymes have been designed to catalyze a wide range of chemical reactions • Ribozymes are naturally occurring catalysts that use metal ions to hydrolyze phosphate ester bonds • Group I and II introns catalyze the excision of introns to produce mature mRNA • ribonuclease P is a nucleoprotein in which the RNA plays the catalytic role and the protein is involved in substrate recognition

Enzyme Catalysis