W31N Enzyme and Biochemistry

• Enzyme Kinetics are being discussed
• Professor Mark Gray is giving this lecture

Steady State Kinetics

• Pre-steady State involves isolated enzyme mixed with substrate, this builds up an enzyme-substrate complex
• Steady states are achieved in microseconds
• Enzymes exist in the steady state most of the time
• The enzymes exist in the steady state in order to regulate metabolism
• In the steady state new material produced = current material destroyed

Assumptions

• Concentration of enzyme is negligible compared to the substrate concentration
• Initial reaction rate is examined, with zero substrate pool depletion and accumulation of products
• V0 (initial reaction rate) is proportional to [E]0 (initial enzyme concentration)
• Measurements are typically taken in the first minute of reaction to be well within the steady-state domain

'Saturation Kinetics'

• V0 follows ‘saturation kinetics’ concerning [S] (substrate concentration)
• At low [S], V0 is proportional [S], meaning the reaction rate is linear in relation to [S]
• At very high [S], V0 approaches Vmax, this is the theoretical maximum rate that the enzyme can operate at

Michaelis–Menten Equation

• The following formula describes the relationship between initial reaction rate (V0), enzyme concentration ([E]0), substrate concentration ([S]), and two constants: kcat (catalytic rate constant) and KM (Michaelis constant).
• V0 = ([E]0[S]kcat) / (KM + [S])
• KM is the Michaelis Constant, it dictates how well you can measure Vmax

Determining KM

• Vmax is the maximum possible velocity that an enzyme can operate
• KM is the substrate concentration which gives a half Vmax

Michaelis–Menten Mechanism

• ES formation (enzyme-substrate) is rapid and reversible
• ES is a non-covalent complex, all the ‘chemistry’ happens in the 2nd step
• kcat is the turnover number, and the step will follow 1st order kinetics

From the mechanism

• In the presence of a substrate you get the following equation = [ES] = [E]0[S]/ Ks + [S]
• This creates the following equation V0 = [E]0[S]kcat/ Ks + [S]
• Where Ks = [E][S]/ [ES], Ks is the equilibrium constant and V0 = kcat[ES]
• Where [E]0 = [E] + [ES]

Comparison of Equations

• KM is often close to Ks, which means that M-M kinetics are operable
• KM > KS means ES dissociation is significant in comparison to kcat (Briggs-Haldane Kinetics).
• KM < KS means that long-lived intermediates exist after substrate binding

Lineweaver-Burk Plot

• It involves a more accurate way of determining Vmax and KM
• Using a double reciprocal graph of measured rate Vo vs substrate concentration [S].
• 1/V0 = KM/Vmax[S] + 1/Vmax
• y = mx + c

Disadvantages of the Double Reciprocal

• Compression of data points occurs with high [S] into a small region
• This favours data with low [S]
• Eadie & Hofstee decided to multiply everything through by V0Vmax, but this isn't perfect

The Eadie-Hofstee plot

• Advantages include all values of [S] are weighted equally and better KM determination
• A disadvantage is that it is time-consuming
• Both axes depend on V0 which can introduce potential for experimental error

The Woolf-Hanes plot

• Terms are multiplied for the L-B plot through [S]
• An advantage is, it is quicker at getting the data than E-H.
• A disadvantage is KM is intercept, this is not slope, and is likely prone to error than E-H

Comparing Catalytic Efficiency

• V0 = [E]0[S]kcat/ KM + [S]
• One can assess/compare catalytic efficiencies of different enzymes using the above equation
• Enzymes possess different kcat and KM values which are relative to their cellular environment

Enzymes

• Enzymes are proteins with catalytic functions
• Enzymes transfer electrons, atoms, or functional groups
• Some enzymes transfer electrons, atoms, or functional groups
• These enzymes are given assigned names and code numbers in accordance to their transfered group type and the used donor/acceptor

Catalysis

• It can be conducted by acid or base
• An acid catalyst can be specific or general

Enthalpy and the hydrophobic effect

• See full text for details on slide*
• It is favourable with an increased enzyme structure

Amino acids

• Amino acid sidechains can play a part in acid or base catalysis

Coenzymes and Cofactors

• Enzymes are proteins with catalytic function, but there are more parts
• Several inorganic elements act as cofactors for enzymes

Premade molecules

• They are often small and organic, which help enzymes catalyse
• EX: Coenzymes that serve as transient carriers of specific atoms or functional groups.

Chymotrypsin

• It is a serine protease, and member of the enzyme family
• The Disulphide linkages: yellow, Catalytic Groups, red.

Peptides and Proteins as Drugs

• Oral administration results in enzymatic hydrolysis of peptide bonds
• Key metabolic enzymes include carboxypeptidases, aminopeptidases
• Issues can be circumvented by Decreasing Proteolysis, such as by:

Decreasing Proteolysis

• Replacing selected L-amino acids but retaining intended activity, or by changing primary-to-secondary amides
• Other options include reversing the peptide bond with pseudo peptides, or co-administration of protease inhibitors

Administration Routes

• Oral administration has very low absorption, needing alternative routes e.g. nasal
• Leuprolide is an example of an analoque of gonadotropin with various rates of absorption

Designing Peptide Drugs

• Peptidomimetics is a practice of mimicry, by fooling a receptor into thinking that it is binding the actual peptide
• Or by using a non-hydrolysable peptide mimic
• Alterations can be by replacement or non-pharmacophoric polar sections
• And peptide backbones can be replaced with alternate atoms / groups

Unnatural Amino Acids

• Unnatural amino acids can be used
• Different structures are possible with different conformational restrictions

Conformation

• Conformation restriction can be done along the peptide backbone
• Mimicry of secondary structure can be applied

RGD Scaffold

• RGD is one letter code for the sequence arginine-glycine-aspartic acid
• The motif is known to block fibrinogen binding to its receptor
• By blocking this binding, it prevents platelet aggregation and valuable treatment for stroke and heart problems

A somatostatin agonist

• Somatostatin (Growth Hormone Inhibiting Hormone) is a 14-residue peptide macrocyclized through a Cys-Cys disulfide bridge
• A glucose scaffold analogue of a small cyclic peptide has proven effective in GH inhibition

Peptidomimetic of Thyrotropin

• This compounds and derivatives lead treatment for Alzheimer's and other cognitive disorder There are many options for changing a peptide backbone i.e. pseudopeptides

Approaches

• Azapeptides, azatides, or peptoids offer new synthetic approaches
• Combinatorial syntheses provide access for many test cases

HIV Protease

• Crucial for formation of mature infectious virions

Natural Targets

• Natural targets block the binding of fibrinogen
• HIV protease is selective for this peptide sequence and this can all be mimicked through design peptide modification

Binding

• Binding can utilize networks including van der Waals forces and aromatic stacking
• Biological processes are regulated by binding events
• These are thermodynamic phenomena, regulated in space
• These regulations are impacted by salt-bridging and hydrogen bonding