Enzyme structure Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase

Enzymes are Catalyst Enzymes speed up chemical reactions. Enzymes are catalyst to over 4000 biochemical reactions! Enzymes are reusable molecules found in living things. During this part of the reaction the molecules are said to be in a transition state. Not permanently changed in the process Are specific Act on substrates

Activation Energy Enzymes work by lowering the activation energy needed to start a chemical reaction. Enzymes work by weakening bonds which lowers activation energy Sugar example http://en.wikipedia.org/wiki/File:Activation_energy.svg

The substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site

The active site One part of an enzyme, the active site, is particularly important The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation energy; this is called the rate-limiting step. In a simple case, the rate-limiting step is the highest-energy point in the diagram for interconversion of S and P.

REGULATION OF ENZYME ACTIVITY Allosteric binding sites: Allosteric enzymes are regulated by molecules called effectors (modifiers) that binds nonconvalently at a site other than the active site. By Covalent Modification: Many enzymes are regulated by covalent modification, most frequently by the addition or removal of ‘phosphate’ group to serine, threonine or tyrosine residue of the enzyme by kinases. (enzyme) Induction and repression of enzyme synthesis: Cells can also regulate the amount of enzymes present by altering the rate of enzyme synthesis.

REGULATION CONT…. 4. Zymogen Cleavage: Some enzyme are synthesized as inactive precursor, called zymogens, that are activated by proteolysis (e.g., digestive enzyme, pepsinogen is inactive and cleaved to pepsin which is active chymotrypsin) 5.Location within the cell: Many enzymes are localized in specific organelles within the cell. This, compartmentation helps in the regulation of the metabolic pathway.

Enzyme enhance chemical resctions by rearrangement of COVALENT bonds and lowering the activation energy (and thereby accelerate the reaction) by providing an alternative, lower-energy reaction path.

2. The second part of the explanation lies in the NON- COVALENT interactions between enzyme and substrate. Much of the energy required to lower activation energies is derived from weak, noncovalent interactions between substrate and enzyme. What really sets enzymes apart from most other catalysts is the formation of a specific ES complex. The interaction between substrate and enzyme in this complex is mediated by the same forces that stabilize protein structure, including hydrogen bonds and hydrophobic and ionic interactions. Formation of each weak interaction in the ES complex is accompanied by release of a small amount of free energy that provides a degree of stability to the interaction. The energy derived from enzyme-substrate interaction is called binding energy, delta GB. Its significance extends beyond a simple stabilization of the enzyme-substrate interaction. Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions.

Cofactors An additional non-protein molecule that is needed by some enzymes to help the reaction Tightly bound cofactors are called prosthetic groups Cofactors that are bound and released easily are called coenzymes Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors

This system divides enzymes into six classes, each with sub- classes, based on the type of reaction catalyzed. Each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it catalyzes. As an example, the formal systematic name of the enzyme catalyzing the reaction ATP:glucose phosphotransferase, which indicates that it catalyzes the transfer of a phosphoryl group from ATP to glucose. Its Enzyme Commission number (E.C. number) is 2.7.1.1. The first number (2) denotes the class name (transferase); the second number (7), the subclass (phosphotransferase); the third number (1), a phosphotransferase with a hydroxyl group as acceptor; and the fourth number (1), D-glucose as the phosphoryl group acceptor. For many enzymes, a trivial name is more commonly used—in this case hexokinase. A complete list and description of the thousands of known enzymes is maintained by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (www.chem.qmul.ac.uk/iubmb/enzyme). Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)

Classification of Enzymes Enzymes are classified according to the type of reaction they catalyze: Class Reactions catalyzed Oxidoreductases Oxidation-reduction Transferases Transfer groups of atoms Hydrolases Hydrolysis Lyases Add atoms/remove atoms to/from a double bond Isomerases Rearrange atoms Ligases Use ATP to combine molecules

Active Site of an Enzyme The active site is a region within an enzyme that fits the shape of substrate molecules Amino acid side-chains align to bind the substrate through H-bonding, salt-bridges, hydrophobic interactions, etc. Products are released when the reaction is complete (they no longer fit well in the active site)

Enzyme Specificity Enzymes have varying degrees of specificity for substrates Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond

Lock-and-Key Model In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site This is an older model, however, and does not work for all enzymes

The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate

The Lock and Key Hypothesis This explains enzyme specificity This explains the loss of activity when enzymes denature © 2007 Paul Billiet ODWS

Induced Fit Model In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis - there is a greater range of substrate specificity This model is more consistent with a wider range of enzymes

The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html This explains the enzymes that can react with a range of substrates of similar types

Isoenzymes Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body - they have slight variations in the amino acid sequences of the subunits of their quaternary structure For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes

Factors affecting Enzymes substrate concentration pH temperature inhibitors

Temperature and Enzyme Activity Enzymes are most active at an optimum temperature (usually 37°C in humans) They show little activity at low temperatures Activity is lost at high temperatures as denaturation occurs

The effect of temperature Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

The effect of temperature For most enzymes the optimum temperature is about 30°C Many are a lot lower, cold water fish will die at 30°C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C

pH and Enzyme Activity Enzymes are most active at optimum pH Amino acids with acidic or basic side-chains have the proper charges when the pH is optimum Activity is lost at low or high pH as tertiary structure is disrupted

Optimum pH for Selected Enzymes Most enzymes of the body have an optimum pH of about 7.4 However, in certain organs, enzymes operate at lower and higher optimum pH values

The effect of pH Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur This change in ionisation will affect the binding of the substrate with the active site.

Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration

Substrate concentration: Enzymic reactions Reaction velocity Substrate concentration Vmax Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too.

Enzyme Concentration and Reaction Rate The rate of reaction increases as enzyme concentration increases (at constant substrate concentration) At higher enzyme concentrations, more enzymes are available to catalyze the reaction (more reactions at once) There is a linear relationship between reaction rate and enzyme concentration (at constant substrate concentration)

Substrate Concentration and Reaction Rate The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate) The relationship between reaction rate and substrate concentration is exponential, and asymptotes (levels off) when the enzyme is saturated

Enzyme activity Enzymes are never expressed in terms of their concentration (as mg or μg etc.), but are expressed only as activities. Enzyme activity = moles of substrate converted to product per unit time. The rate of appearance of product or the rate of disappearance of substrate Test the absorbance: spectrophotometer

Enzyme Kinetics

Enzyme Catalyzed Reactions When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed: E + S  ES Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES  E + P The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute The overall reaction for the conversion of substrate to product can be written as follows: E + S  ES  E + P

Enzyme velocity Enzyme activity is commonly expressed by the intial rate (V0) of the reaction being catalyzed. Enzyme activity = moles of substrate converted to product per unit time.

Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions Studying the effects of substrate concentration is complicated—as-- [S] changes during the course of an in vitro reaction as substrate is converted to product. One simplifying approach in kinetics experiments is to measure the initial rate (or initial velocity), designated V0,when [S] is much greater than the concentration of enzyme, [E].

In a typical reaction, the enzyme may be present in nanomolar quantities, whereas [S] may be five or six orders of magnitude higher. If only the beginning of the reaction is monitored (often the first 60 seconds or less), changes in [S] can be limited to a few percent, and [S] can be regarded as constant. V0 can then be explored as a function of [S], which is adjusted by the investigator. At relatively low concentrations of substrate, V0 increases almost linearly with an increase in [S]. At higher substrate concentrations, V0 increases by smaller and smaller amounts in response to increases in [S]. Finally, a point is reached beyond which increases in V0 are vanishingly small as [S] increases. This plateau-like V0 region is close to the maximum velocity, Vmax.

The ES complex is the key to understanding this kinetic behavior This idea was expanded into a general theory of enzyme action, particularly by Leonor Michaelis and Maud Menten in 1913. They postulated that the enzyme first combines reversibly with substrate Because the slower second reaction (Eqn 6–8) must limit the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step, that is, ES.

At any given instant in an enzyme-catalyzed reaction, the enzyme exists in two forms, the free or uncombined form E and the combined form ES. At low [S], most of the enzyme is in the uncombined form E. Here, the rate is proportional to [S] because the equilibrium of Equation 6–7 is pushed toward formation of more ES as [S] increases. The maximum initial rate of the catalyzed reaction (Vmax) is observed when virtually all the enzyme is present as the ES complex and [E] is vanishingly small. Under these conditions, the enzyme is “saturated” with its substrate, so that further increases in [S] have no effect on rate. After the ES complex breaks down to yield the product P, the enzyme is free to catalyze reaction of another molecule of substrate. The saturation effect is a distinguishing characteristic of enzymatic catalysts and is responsible for the plateau this is sometimes referred to as saturation kinetics. When the enzyme is first mixed with a large excess of substrate, there is an initial period, the pre–steady state, during which the concentration of ES builds up. This period is usually too short to be easily observed, lasting just microseconds. The reaction quickly achieves a steady state in which [ES] (and the concentrations of any other intermediates remains approximately constant over time. The concept of a steady state was introduced by G. E. Briggs and Haldane in 1925. The measured V0 generally reflects the steady state, even though V0 is limited to the early part of the reaction, and analysis of these initial rates is referred to as steady-state kinetics. The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed Quantitatively The curve expressing the relationship between [S] and V0 has the same general shape for most enzymes (it approaches a rectangular hyperbola), which can be expressed algebraically by the Michaelis-Menten

Michaelis-Menten equation 1. Michaelis-Menten equation describes how reaction velocity (V) varies with substrate concentration [S]. The following equation is obtained after suitable algebraic manipulation. [S] [S] + KM V = Vmax Note: V means V0 Km: Michaelis constant Km = (k2 + k3)/k1

Enzyme Inhibitors Chemicals that prevent the enzyme from working. Inhibitors decrease the enzyme reaction rate. http://en.wikipedia.org/wiki/File:Competitive_inhibition.svg

Inhibitors (I) are molecules that cause a loss of enzyme activity Enzyme Inhibitors Inhibitors (I) are molecules that cause a loss of enzyme activity They prevent substrates from fitting into the active site of the enzyme: Some enzyme inhibitors are normal body metabolites. Other may be foreign substances,such as drugs or toxins. E + S  ES  E + P E + I  EI  no P formed

Inhibitors Inhibitors are chemicals that reduce the rate of enzymic reactions. The are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system.

The effect of enzyme inhibition Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.

The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are three categories.

Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. A competitive inhibitor is reversible and has a structure like the substrate - it competes with the substrate for the active site - its effect is reversed by increasing substrate concentration Example Malonate is a competitive inhibitor of succinate dehydrogenase - it has a structure that is similar to succinate - inhibition can be reversed by adding succinate

The effect of enzyme inhibition Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples Cyanide combines with the Iron in the enzymes cytochrome oxidase. Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA.

Reversible Inhibitors (Noncompetitive Inhibition) A noncompetitive inhibitor has a structure that is different than that of the substrate - it binds to an allosteric site rather than to the active site - it distorts the shape of the enzyme, which alters the shape of the active site and prevents the binding of the substrate The effect can not be reversed by adding more substrate

Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants

Enzyme Activators X Chemicals that help the enzyme work. Activators increase the enzyme reaction rate. X Activator Substrate Binding Site Active