Enzyme reactions and kinetics

• Enzymes elicit

  a long term change : genetic changes

  a short term change : phosphorylation/dephosphorylation
• Enzymatic nomenclature usually reflects the reaction that the enzyme catalyses

  1. Oxidoreductases - enhances redox reaction
  2. Transferases - Enhances transfer of group/atom between molecules
  3. Hydrolases - Enhances hydrolysis reactions
  4. Lysases - Enhances rate of addition of one group to a double bond or creation of double bond locally
  5. Isomerases - Enhaces rate of isomerisation reactions
  6. Ligase/Synthetase - Enhances rate of joining two molecules together

  Can also be given an EC number - 4 digit code (identification number)
• • Increase in temperature increases the thermal energy of the substrate molecules - more molecules have enough energy to get over the activation barrier
  • As temperature increases, the enzyme structure changes via breaking of weak bonds/interactions that hold the 3D structure - the enzyme unfolds to the point of denaturing which makes it lose its activity
  • A slight increase in temperature helps the enzyme, too much and the enzyme is hindered hence the optimum temperature
• • pH can affect enzyme catalysed reactions via changes in the charge of ionic bonding and reducing enzyme activity outside of the optimal pH
  • Large pH changes will denature the enzyme
• • The active site is where the substrate binds and is converted to a product
  • The protein has a 3D structure and the active site is a small cleft or crevice on the surface
  • The active site is usually non polar to help with the substrate binding (charged groups make binding or release, more challenging)
  • Weak binding forces are better - electrostatic interactions, hydrogen bonds, VDW force and hydrophobic interactions
  • Once the enzyme-substrate complex has formed, the active residues within the active site act on the substrate to convert it to the product
  • This product is then released and the enzyme can bind again
• Induced fit model (Koshland 1958)

  • The binding of a substrate to an enzyme induces a conformational change in the active site
  • The enzyme may change its shape or change the shape of the substrate to promote the reaction e.g. hexokinase with glucose adding a phosphate group
  • Most enzymes show features of lock and key model and induced fit model
• Prosthetic groups

  • If a metal ion is covalently linked to the enzyme (Zn and Fe)
  • The complete enzyme with the prosthetic group is the holoenzyme
  • The enzyme on its own without the prosthetic group is the apoenzyme
• Since absorbance is proportional to concentration, the rate of change in absorbance is proportional to the rate of enzyme activity
• Coenzymes

  NAD+ - catabolic

  NADP+ - anabolic

  The nicotinamide ring is the reactive part of the coenzyme

  FAD and FMN are used in fat metabolism and the flavin mononucleotide unit is the reactive part
• Other cofactors include metal ions (Zn and Fe) - divalent cations

  If the metal ion is linked covalently to the enzyme, it's called the prosthetic group (so haemoglobin has an iron group covalently linked which is a prosthetic group)

  The complete enzyme with the prosthetic group is the holoenzyme

  The enzyme on its own without the prosthetic group is the apoenzyme
• Isoenzymes are different forms of an enzyme which catalyse the same reaction but have different physical or kinetic properties

  Isoenzymes are produced by different genes and come from different tissues

  LDH is an example of this with 5 different isoenzymes - its a tetramer of 2 different subtypes which give the 5 different isoenzymes
• The amount of enzyme present can be determined by measuring its catalytic effect via an assay

  The recognised unit of enzyme activity is the micromoles of substrate converted per unit time

  If the serum is present in a tissue sample, then the activity is expressed as specific activity

  Purified powder - activity of enzyme per minute per milligram (Micromol/ min/ mg)

  In serum - activity of enzyme per minute per litre (micromol/min/l)
• Rate of enzyme reaction can be easily followed - substrate or product which absorbs light at a specific wavelength measured using a spectrophotometer

  Since absorbance is proportional to concentration, the rate of change in absorbance is proportional to the rate of enzyme activity - Beer Lambert Law
• In order to measure the activity of the first enzyme you need :

  • second enzyme and cofactors to be in excess

  Ensures the second reaction is never rate limiting and the first reaction is always the rate limiting reaction
• Most biochemical reactions need to overcome a specific reaction energy barrier to allow the reaction to proceed - no enzyme means the activation energy required is higher
• Enzymes work by stabilising the transition state (high energy and unstable)

  This is done by decreasing the free energy so that less energy is needed to allow the reaction to occur in the presence of the associated enzyme

  This does not change the energy levels of the substrate or product but does increase the rate of reaction as less free energy is needed to pass through the transition state

  The enzyme does not change the overall energy of the reaction
• The rate of many enzymes are altered by the presence of other molecules (known as effectors) that can be activators or inhibitors

  In metabolic pathways a common control mechanism is the inhibition of an enzyme early in a pathway by the end product in a pathway

  This is called feedback inhibition and prevents build up of large quantities of unwanted metabolic intermediates and conserves energy
• Allosteric enzymes are often multi subunit proteins with one or more active sites

  In allosteric enzymes the binding of substrate to one active site affects the binding of substrate molecules to other active sites

  The active sites are said to behave cooperatively with respect to substrate binding and catalysis - one subunit is bound so the others shouldn't OR one subunit is bound so the others bind more
• Binding of the substrate to one active site induces a conformational change in the protein that is transmitted to the other active sites and alters their substrate affinity

  This means allosteric enzymes are particularly sensitive to small changes in substrate concentration within the physiological range - more efficient at dealing with small substrate concentrations and using them effectively
• Monod Wyman Changeaux Model

  In this model the subunits of the allosteric protein or enzyme can only exist in one of two states : T or R

  T state subunits are in a tense state - these are compact and relatively inactive - when no substrate is present

  R state subunits are relaxed - expanded and active with high substrate affinity - equilibrium shifts to the R state upon binding

  No intermediate states allowed - so if 1 subunit changes conformation, then they all change together - no mixed oligomers with both T and R states
• Koshland sequential model

  In this model the change in structure is transmitted through the protein one subunit at a time as the active sites are filled

  The binding of one subunit can influence the affinity of binding to another subunit without inducing a transition in the whole protein

  The subunits change in turn rather than all at once

  These changes are dependent in the degree of coupling or physical interaction between the subunits

  In the model enzyme substrate binding affinity varies with the number of bound substrate molecules
• Other modifications

  Reversible covalent modifications - addition or removal of a phosphate group - kinases and phosphatases in metabolic pathways operate in this manner

  Proteolytic activation - protein exists as a larger inactive form of the enzyme (proenzyme) - activated by hydrolysis of a peptide bond e.g. trypsin and chymotrypsin as well as the coagulation cascade
• The rate of an enzyme catalysed reaction is its velocity - normally reported at the start of the reaction (t0) as the rate is normally fastest here - substrate concentration is highest and no product has been formed so there's no feedback inhibition

  This starting velocity is called the initial velocity and is denoted by the symbol V0 - is measured before around 10% of the substrate has been used up and the units of it are micromol/min
• Initial velocity is obtained by drawing a straight line through the linear part of the curve starting at the 0

  The slope of this line is the V0

  The maximum rate of the reaction is the maximum velocity and is denoted by Vmax - this is when the enzyme is saturated with substrate so the reaction can't be any faster no matter how much substrate is present
• When thinking about product formed in an enzyme catalysed reaction, the inital (linear) part of the graph shows rapid product formation

  This then plateaus because substrate gets used up and the enzyme is working as fast as it can because there's a backlog of substrate to work through with a finite amount of active sites