Trypsin: a case study

Meggy Phils #3

Trypsins
Family members have different substrate specificities on carboxyl side of peptide bond to be cleaved: chymotrypsin has large hydrophobic substrates (Y, W, P) elastase has small hydrophobic (G, A, V), trypsin has charged R/Ks. Trypsin family proteins are synthesized as proenzymes and activated by proteases.

Mechanism
Wont go over electron pushing here. Mechanism has two steps: Acylation: enzyme forms carboxylic ester linkage to C of peptide bond. This causes peptide bond cleavage, releasing peptide P1 Deacylation: water substitutes for ester bond, releasing peptide P2 and enzyme.

Kinetic Scheme

The speed of the reaction can be determined by the rate of substrate binding, or acylation, or deacylation. The slowest one will be the bottleneck that determines how quickly the protease can cleave peptides. Rate of ES complex formation is not discussed, just acylation/deacylation.

Proof for the Trypsin mechanism

If acylation is the rate-determining step, changes in reaction rate will be related to changes in rate of acylation Perform assays of activity using substrates predicted to form acyl complex at different rates Even though rates of acylation would be different using different ester substrates, rate of overall trypsin reaction does not change: as shown on Kinetics of a common intermediate slide, esters of N-acetyl-L-tryptophan derivatives have very similar Kcat.

Burst Phase kinetics are observed

Different assay which produces P-nitrophenolate (yellow substance can be monitored spectrophotometrically) as P1, product of acylation Can observe differences between acylation and deacylation rates in biphasic enzyme activity graph Burst phase while first round of very fast acetylation occurs Slows down as enzymes are occupied by the need to free themselves of P2 in slower deacetylation reaction

Specificity
Next couple slides are about role of active site residues- I will skip to specificity expts. Graf/Hedstrom mutated residues in the substrate binding pocket to see if they could change trypsin specificity to that of chymotrypsin. Needed to also mutate residues on loops near the binding site to get activity Still only worked on long substrates Although not rate-determining when the substrate is correct the acylation step does determine specificity i.e acylation will be very slow for poor substrates (table of k2 and k3, second to last slide)