Enzymes II

• Measure the rate at which E + S makes E + P • Alter the viscosity - Add glycerol to mixture, viscosity increases, diffusion is reduced. Does a change in viscosity (therefore velocity) slow the reaction rate? • If so, the enzyme is diffusion limited and catalytically perfect. e.g. Carbonic anhydrase

• Divide Kcat / Km (Or use the diffusion-controlled rate of encounter, 10^8 M-1S-1) • Is the enzyme reaction rate for E + S -> E + P close to the diffusion-controlled rate of encounter?

• Determine the actual free energy profile of the enzyme reaction. Are the diffusion steps rate limiting?

• Catalyses conversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (3 carbon intermediates in glycolysis) • Makes it so that instead of there being a big energy change and conversion of A to Z, it happens via intermediates that don’t have a high energy change between them, easing the conversion. • Reaction is limited by E + S (substrate diffusion into enzyme), so decreasing energy levels doesn’t increase rate of reaction.

Hydrolyse peptide bonds Serine, cysteine, aspartyl, metallo-proteinases

Chymotrypsin, trypsin

They have a very reactive serine at their active site

If the residue of the terminal is hydrophobic (Phe, Trp, Tyr)

If the residue of the terminal is positively charged (Lys, Arg)

If the residue of the terminal is narrow/small

Chymotrypsin has a hydrophobic pocket which fits with the hydrophobic side chain

Trypsin binding pocket has an Asp molecule which has a negative charge, producing an electrostatic interaction with the residue

Binding pocket for residue in elastase is narrow, blocked off by some valine residues, so it fits best with small residues.

Very high free energy of activation for hydrolysis (without an enzyme)

• Water attacks carbon atom of ketone of amide group in polyp chain • Lone pair of electrons of oxygen help form tetrahedron intermediate, giving the O bound to the carbon a negative charge. • The intermediate collapses, breaking the peptide bond and releasing the carboxyl group and the amide.

Different reaction pathway to uncatalysed reaction, using Ser-OH group in the active site of every serine protease.

• Ser-OH attacks the carbon of the ketone of the peptide bond • Again tetrahedron intermediate formed and collapses. • An ester is instead formed, an acyl enzyme intermediate with an ester bond to the polypeptide chain (Acylation)

• Water comes in and attacks the acyl enzyme intermediate, forming a tetrahedral intermediate • The tetrahedral intermediate then collapses, to release the enzyme and restore to its original state • Carboxyl group and amide group released.

Within the active site of the serine OH, the serine is actually forming a hydrogen bonded network with the side chains of two other residues (histadine and aspartate) This network forms a charge relay system, which pulls the proton off the serine OH and onto

Within the active site of the serine OH, the serine is actually forming a hydrogen bonded network with the side chains of two other residues (histidine and aspartate) This network forms a charge relay system, which pulls the proton off the serine OH and onto the imidazole group, then to the aspartate. The proton is shifted away from the oxygen, making the oxygen very electronegative. Easier to carry out nucleophilic attack.

Synthesises ATP - the last step in oxidative phosphorylation. Driven by protons

Protons (low on the inside, high on the outside, forming gradient)

Lollipop shape Stator with 6 sub units, hole down the middle held in place by proteins Rotating spindle in hole Spindle in contact with 3 active sites, where ATP is made Protons rotate rotor, registers the active sites to make ATP.

• ADP and phosphate are bound to an active site • The rotor rotates 120 degrees • Provides energy to release previously synthesised ATP in another active site of the enzyme • This squeezes the ADP and the phosphate, to make ATP at the other active site • This continues in a loop.