Catalytic Mechanism of Enzyme Reaction Remarkable properties of Enzymes as catalysts 1. Catalytic power 2. Specificity 3. Regulation Ad1. Catalytic Power - enzymes increase the rate of reaction by as much as 1012 –fold - not many examples to compare between the rates of an enzyme-catalyzed reaction and the reaction occurring under similar conditions (temperature, pH etc) but in the absence of enzyme as it is too low to be measured - Comparison between enzymatic and non- enzymatic catalysts also difficult. As enzymatic catalysts occur : o Much higher rates o Lower temperature It is well illustrated by the process of nitrogen fixation (N2 ammonia) catalyzes by nitrogenase complex (300 K ,pH neutral) Industrial synthesis of ammonia from nitrogen and hydrogen temp 700-900 K, pressure 100- 900 atm, with iron catalyst Ad2. Specificity - enzyme highly specific both in : o nature of substrate o the reaction they catalyze - the range of specificity varies between enzymes o low specificity the specificity base on bond specificity ex. Peptidase, phoshatase, esterase utilize a wide range of substrates which contain the required chemical bond mostly for degradative enzymes but not biosynthetic enzymes. o intermediate specificity group specificity ex. Hexokinase catalyse the phosphorylation of a variety of sugars provided they are aldohexose o absolute / near absolute specificity. only catalyse a reaction with a single substrate at an appreciable rate Ad3. Regulation - The catalytic activity regulated by small ions or other molecules - Ex. The breakdown of glycogen in skeletal muscle Carbohydrate reserved degraded to generate ATP required for muscle contraction The muscle contraction is triggered by release of Ca2+ from the sarcoplasmic reticulum also ensure the continuation of ATP production Feedback inhibition phenomena common in many biosynthetic pathway Ex. Biosynthetic pathway leading to the synthesis of pyrimidine nucleotide the end product UTP and CTP block the first enzyme they are able to to limit the flow of metabolites into the pathway and regulate their own biosynthesis HOW ENZYME WORKING A catalyst works simply by lowering the energy barrier of a reaction, ΔGº± Catalysts provide an alternative way the catalyst increases the fraction of molecules that have enough energy to attain the transition state, thus making the reaction go faster in both directions. The position of the equilibrium (the amount of product versus reactant) is unchanged by a catalyst. Catalysts lower the energy barrier in two ways: o The catalyst binds a substrate in an intermediate conformation that resembles the transition state, but has a lower energy. lead to multiple intermediate states that bypass the transition state. An intermediate state is a metastable state of a molecule. o In a non-catalyzed reaction the entropy may be highly negative due to the highly specific orientation required in order for a reaction to occur. Catalysts can lower the negative entropy by binding reacting molecules only in the proper mutual orientation, thus increasing their reactivity E + S ↔ ES ↔ EP ↔ E + P Collision Theory Molecular velocity determines the binding of enzyme and substrate, thereby determine ES formation and Enzyme catalytic velocity Transient ES complex undergo INTRA-Molecular straining decreasing initial energy requirement for catalytic reaction to produce reaction product The highest point of the energy profile is designated the transition state of the reaction In all cases, the catalyst does not cause a shift in the equilibrium between reactant (s) and product (s) only increases the rate at which that equilibrium is attained. Various factors leading to the rate enhancements a. proximity and orientation effects b. acid-base catalyst c. covalent catalyst d. strain or distortion e. changes in environment ad.a proximity and orientation effects commonly an enzyme could increase the rate of reaction involving more than one substrate by binding the substrates at adjacent sites and therefore cringing them into close proximity with each other so reaction occur more readily Orientation with respect to each other influence the rate of reaction Enzymes make sure that the reactants are in the correct orientation as they approach each other Example In this examples the restrictions placed on rotation of single bonds by bridge structure ensures that the preferred orientation of the reacting groups closely resembles that of the transition state of the reaction Less rotational entropy (degrees of freedom) occurs as the reaction proceed towards the transition state The smaller negative entropy of activation lead to an increase in the rate of reaction Adb. Acid-Base catalyst Many reactions of the type catalyzed by enzymes are known to be catalyzed by acids and/or base Since enzymes contain a number of amino acid side chains which are capable of acting as proton donors or acceptors acid-base catalysis important Adc. Covalent catalysis (intermediate formation) Reactions can be speeded up by the formation of intermediates Many of the examples of covalent catalysis in enzyme-catalyzed reactions involve attack by a nucleophilic side chain at electron-deficient centre in the substrate nucleophilic catalysis Ad.d Strain or distortion A substrate may be distorted on binding to the appropriate enzyme speed up the reaction if the distortion lowered the free energy of activation by making the geometry and electronic structure of the substrate more closely resemble the transition state Strain also give a stabilization of the transition state of the reaction Enzymes make favorable contact with the transition state of the substrate Ad5. changes in environment The rates of many organic reactions are highly sensitive to the nature of of the solvents in which they occur ENZYME MODEL The induced fit model of enzyme action is a modification of the lock-and-key model originally proposed by Emil Fischer in 1894 The lock-and-key model proposes that an Enzyme/substrate pair is like a lock and key. Though it explains the specificity of enzyme /substrate pairs, it does little to explain catalysis, because a lock does not change a key the way an enzyme changes a substrate. In 1958, Daniel Koshland proposed the induced fit model to explain enzymatic catalysis proposed that distortion of the enzyme and the substrate is an important event in catalysis. Enzymes do more than simply bind and position substrates, however. Enzymes 1. Bind substrate(s); 2. Lower the energy of the transition state; and 3. Directly promote the catalytic event occur as a result of specific amino acid side chains that physically interact with the substrate and end up promoting the reaction. When the catalytic process is complete, the enzyme must be able to release the product or products and return to its original state for another round of catalysis. Triose Phosphate Isomerase Catalysis Triose phosphate isomerase catalyzes the following reaction: Glyceraldehyde-3-Phosphate (G3P) <=> cis- enediol intermediate <=> Dihydroxyacetone Phosphate (DHAP) The active enzyme is a dimer of two identical subunits. The active site (the place on the enzyme where catalysis occurs) can accommodate either G3P or DHAP At the active site, a glutamate residue (Glu 165) and a histidine (His 95) are essential for function of the enzyme. The reaction steps : E + G3P <=> E-G3P (Binding of G3P) E-G3P <=> E-ed (Conversion to enediol) E-ed <=> E-DHAP (Conversion to DHAP) E-DHAP <=> E + DHAP (Release of DHAP) Like other enzymes, triose phosphate isomerase lowers the energy barriers of the transition States Both triose phosphate isomerase and serine proteases have a histidine and an acidic residue in their active site. Histidine is very common in active sites, because it readily accepts or donates protons at physiological pH. Multisubstrate Reactions Most biochemical reactions involve two or more substrates, often resulting in multiple products. An example is proteolysis, which involves two substrates (the polypeptide and water) and two products (the two fragments of the cleaved polypeptide chain). When an enzyme binds two or more substrates and releases multiple products, the order of the steps becomes an important feature of the enzyme mechanism. Several classes of mechanisms include the following: o Random substrate binding o Ordered substrate binding o The "ping-pong" mechanism Ad. Random substrate binding In this mechanism, either substrate can be bound first, though in many cases one substrate will be favored for initial binding, and its binding may promote the binding of the other. Ad. Ordered substrate binding This mechanism occurs when one substrate must bind before a second one can bind significantly. often observed in oxidations of substrates by the coenzyme nicotinamide adenine dinucleotide (NAD+). Ad. The ping-pong mechanism This occurs when a catalytic sequence of events occurs, such as one substrate is bound, one product is released, a second substrate is bound, and a second product is released.