Three-Point Binding Model First proposed by Ogsten (1948) to explain biological enantioselection/enantiospecificity Serves as a model for chromatographic chiral stationary phases Preferential binding occurs via intramolecular non-covalent forces: H-bonding salt bridge Ionic Dipole-dipole Van der Waals

CH 2 OH moieties are different because of non-equivalent binding sites in the enzyme Enantioselection by an Enzyme

Three-Point Binding Model - Enantiospecificity Only one enantiomer binds to enzyme & is involved in reaction

With the other enantiomer…

 we get enantiospecificity (substrate & biomolecule are chiral) To do this efficiently, we need a large biomolecule to align three binding sites to give high specificity One problem with model: –Model is a static representation → “lock & key”

Binding The cost of binding: K m (Michaelis constant): small value indicates high affinity for substrate  K binding ( ~ 1/K m ) Strong binding → K > 1 ΔG= -RT ln K ΔG must be –ve

ΔG binding = ΔH binding - TΔS binding For 2 molecules in, 1 out: ΔS is –ve  (-TΔS) term is +ve  Entropy disfavors binding of substrate to enzyme  To get good binding, need –ve ΔH (i.e. bond formation) Each non-covalent interaction is small (H-bond ~ 5 kcal/mol), but still gives a –ve ΔH Enzymes use many FG’s to sum up many weak non- covalent interactions (i.e. 3 points)

Back to tyrosyl-tRNA synthase:

Tyrosyl-tRNA synthase Use binding to orient CO 2 - nucleophile adjacent to P  specifically as electrophile → specificity Many non-covalent interactions overcome entropy of binding: H-bonds Can isolate this complex in the absence of t RNA

Tyrosyl-tRNA Synthase. tyr-adenylate

* * * * Main chain contacts Tyr specificity Binding AAs 3 point binding enantiospecificity Bind ATP ATP, not dATP

* * * * Main chain contacts Orient  PO 4 towards CO 2 - Increase P  +

We have examined the crystal structure of tyrosyl-tRNA synthase (Tyr & ATP bound) –Key contacts –3 point binding model for (S)-tyrosine We inferred geometry of bound ATP prior to reaction (i.e. ATP is no longer bound to enzyme) Step 1: CO 2 - attacks PO 4 2- (  ) giving pentacoordinate P (trigonal bipyramidal) intermediate

Step 2: Diphosphate must leave Cannot “see” this step  PP i has already left the enzyme site in the crystal structure However, can use model building to include P  & P  of ATP: Thr40 & His45 form H-bonds to P     **Stronger H- bonds are formed in TS than in trig. Bipyramidal intermediate Lower TS energy  accelerate collapse of intermediate Gln195

Tests of Mechanism 1)Site-directed mutagenesis –Replace Gln195 with Gly  (Gln195Gly) Rate slows by > 1000 fold ΔΔG  ~ 4 kcal/mol Developing -ve charge (on oxygen) in TS is no longer stabilized Energy diagram? Other mutants: –Tyr34Phe –His48Gly –These other mutations showed smaller decreases in ΔG –All contribute in some way to stabilize TS

2)Do Thr-40 & His-45 really bind  /  phosphates? Thr 40  Ala (  7000 fold) His 45  Gly (  300 fold)  Both decelerate the reaction Double mutant  300,000 fold slower!

A Chemical Model for Adenylate Reaction Mimic the proximity effect in an enzyme with small organic molecules: Detect by UV Rate is comparable to tyrosyl-adenylate formation  unimolecular reaction

Step 3: 3’-OH attacks acyl adenylate -ve charge increases on O of carbonyl  H- bonding stabilizes this charge (more in TS than in SM)  H-bonding (of Gln) is “more important” for TS Step 2 leads to adenylate; CO 2 H group is now activated Once activated, tRNAtyr-OH can bind

X-ray Structure of tRNA Gln Example of tRNA bound to tRNA synthase (stable without Gln) tRNA (red) binds to enzyme via multiple H-bonds 3’-OH oriented close to ATP (consistent with proposed mechanism in tyrosyl-tRNA) 3-’OH ATP

Unique Role of Methionine Recall, Methionine is the 1 st amino acid in a peptide/protein (start codon) As seen previously, Met is also formylated From N-formyltetrahydrofolate protected

Protection with formyl group allows condensation one way around only (only one nucleophile) Reaction is catalysed by becoming pseud- intramolecular (recall DNA template synthesis): Ribosome holds pieces together  Ribosome is cellular “workbench” tRNA fMet falls off P site Dipeptide moves over to P site

Control of Sequence mRNA (messenger RNA) made by copying sequence of DNA in gene Goes to ribosome, along with rRNA (ribosomal RNA-part of ribosome structure) & tRNA (with AAs attached) In mRNA, 3 nucleotides of specific sequence encode 1 amino acid (CODON) R-tRNA R has 3 nucleotides complementary through base pairing to the codon for R Specific binding at A site Codons for start & stop control the final protein length

CODON Met Tyr P site A site Rxn & translocation Met Tyr Arg P site A site Met Tyr Arg

Catalysis of Reaction? Synthesis on ribosome is faster by 10 7 than rxn without ribosome Peptide formation is not catalyzed by protein → no protein within 20 Ǻ of “active site” rRNA (catalytic RNA) has been proposed : Adenosine from rRNA

However, modification of bases has shown little effect on catalytic activity (2-fold decrease) May be the 2’-OH (of tRNA) at last nucleotide on P site: i.e., the substrate! (see Nature Struct. Mol. Biol. (2004), 11, p 1101 Modified sugar at 3’OH: OH → H OH → F Both substitutions reduce rate by 10 6 !

adenosine

Why the Reduction in the Rate? Accounts for most of rate acceleration  e.g. of catalytic RNA & substrate catalysis P siteA site