Enzyme Kinetics Flashcards
What is an Enzyme?
A substance (usually a protein) that increases the rate of a chemical reaction without itself being changed in the overall process.
What is Primary, Scondary, Tertiary and Quarternary Structure?
- Primary Protein Structure: Sequence of chain of amino acids
- Secondary Protein Structure: Hydrogen bonding of peptide backbone caues the amino acids to fold into a repeating pattern
- Tertiary Protein Structure: Three-dimensional folding pattern of protein to side chain interactions
- Quarternary Protein Structure: Prtein consiting of more than one amino acid chain
What are characterisitics of Enzymes?
- Enzymes increase the rate of a reaction
- Enzymes do not change the reaction equilibrium
- Enzymes are specific for their substrates
- Enzymes are regulated: Genetic (transcription/translation) & Allosteric
Why do labs measure enzymes?
- During disease, tissue damage may lead to release of enzymes from tissues: e.g Release of aminotransferases (ALT and AST) indicate liver damage
- Inborn errors of metabolism may be due to a deficiency of a particular enzyme: e.g PKU, due to deficiency of the enzyme phenylalanine hydroxylase.
Which factors affect enzymatic activity?
- The rate of an enzyme-catalysed reaction is proportional to the concentration of the enzyme.
- pH
- Temperature
- Type of buffer
- [Cofactor]
- [Substrate]
- Incubation time
- [Enzyme]
What are enzymatic rate measurement methods?
Fixed-time method: use enzymes with high affinities (low Km)
- Measure substrate or product concentration at two time points and calculate the difference then divide by time period to obtain rate.
Continuous monitoring method: use enzymes with low affinities (high Km).
- Reaction is monitored by continuously taking a rate measurement over a designated time period.
What are Units for Expressing enzymatic activity?
-
SI unit of activity – katal
- The amount of enzyme which will convert 1 mole of substrate in 1 second.
-
International unit – U
- The amount of enzyme which will convert 1 μmole of substrate in 1 Minute
What are steps involved in calculating enzyme activity?
- Convert physical measurement (e.g. absorbance) made over a timed interval:
- Divide change in absorbance by the time period of measurement(minutes) to give rate (ΔA/min).
- Convert into substrate concentration units and derive rate for the enzyme-catalysed reaction: Divide by molar absorptivity (L/mol/cm) and cuvette path length (cm): [Beer-Lambert’s Law]
- Convert to the concentration of enzyme units in the clinical sample: Multiply by 1,000,000 to convert from mol to μmol. Multiply by total reaction volume (mL) and divide by sample volume(mL) – ‘dilution factor’
What is the final equation for enzyme activity?
(ΔA/min x 1,000,000 x Total vol (mL)) / ε (L/mol/cm) x l (cm) x Sample vol (mL)
What is the function of enzymes?
- Enzymes accelerate the approach to equilibrium between substrates and products by lowering the activation energy of the reaction.
- No effect on position of equilibrium
- The ‘standard free energy of activation’ (ΔG°‡) is the additional free energy that substrate molecules must have to attain the transition state.
What is the Michaelis-Menten Equation?
- Describes effect of substrate concentration on rate of enzyme catalysed reactions.
E + S ⇌ ES ⇌ E + P
- A few milliseconds after mixing enzyme (E) and substrate (S) the enzyme-substrate complex [ES] builds up and does not change
- This is called the Steady State [ES] is constant)
What is Michaelis Menten Equation?
V = (Vmax [S]) / Km + [S]
v = initial reaction velocity at [S]
KM = the Michaelis constant
Vmax = the maximum possible initial reaction velocity
What is Km?
- Km is the substrate concentration at half-maximal velocity
- Km is a measure of the strength of the ES complex:
- A high Km indicates weak binding; a low Km indicates strong binding
What happens at low concetrations of subtrate to enzymatic kinetics?
- At low concentrations of substrate [S], the initial velocity of the reaction (v) is directly proportional to the substrate concentration.
- The equation approximates to v = Vmax [S] / Km which is a linear expression.
- When reaction rate is proportional to a single concentration term it is said to follow first-order kinetics.
What happens at high concetrations of subtrate to the enzymatic kinetics?
- At very high [S], for example when [S] >>> Km most of the enzyme exists as the ES complex.
- The enzyme is saturated with substrate.
- The equation approximates to v = Vmax [S] / [S] = Vmax
- Rate becomes independent of [S] and constant (v = Vmax)
- When reaction rate is independent of [S] it is said to follow zero-order kinetics.
