ENZYMES A protein with catalytic properties due to its power of specific activation © 2007 Paul Billiet ODWS

Chemical reactions Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY During this part of the reaction the molecules are said to be in a transition state. © 2007 Paul Billiet ODWS

Reaction pathway © 2007 Paul Billiet ODWS

Making reactions go faster Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway “a short cut” © 2007 Paul Billiet ODWS

An enzyme controlled pathway Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions. © 2007 Paul Billiet ODWS

Enzyme structure Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase © Dr. Anjuman Begum © 2007 Paul Billiet ODWS

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 © H.PELLETIER, M.R.SAWAYA ProNuC Database © 2007 Paul Billiet ODWS

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 Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997) © 2007 Paul Billiet ODWS

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 © 2007 Paul Billiet ODWS

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 © 2007 Paul Billiet ODWS

The Lock and Key Hypothesis Enzyme may be used again Enzyme-substrate complex E S P Reaction coordinate © 2007 Paul Billiet ODWS

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

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) © 2007 Paul Billiet ODWS

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 © 2007 Paul Billiet ODWS

Factors affecting Enzymes substrate concentration pH temperature inhibitors © 2007 Paul Billiet ODWS

Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11 © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

The effect of temperature Temperature / °C Enzyme activity 10 20 30 40 50 Q10 Denaturation © 2007 Paul Billiet ODWS

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 © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories. © 2007 Paul Billiet ODWS

The effect of enzyme inhibition 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. Enzyme inhibitor complex Reversible reaction E + I EI © 2007 Paul Billiet ODWS

The effect of enzyme inhibition Succinate Fumarate + 2H++ 2e- Succinate dehydrogenase CH2COOH CHCOOH COOH CH2 Malonate © 2007 Paul Billiet ODWS

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. © 2007 Paul Billiet ODWS

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 © 2007 Paul Billiet ODWS

Classification of enzymes. Units of enzyme activity. 31

Naming of Enzymes Common names are formed by adding the suffix –ase to the name of substrate Example: - tyrosinase catalyzes oxidation of tyrosine; - cellulase catalyzes the hydrolysis of cellulose Common names don’t describe the chemistry of the reaction Trivial names Example: pepsin, catalase, trypsin. Don’t give information about the substrate, product or chemistry of the reaction 32

Principle of the international classification All enzymes are classified into six categories according to the type of reaction they catalyze Each enzyme has an official international name ending in –ase Each enzyme has classification number consisting of four digits: EC: 2.3.4.2 First digit refers to a class of enzyme, second -to a subclass, third – to a subsubclass, and fourth means the ordinal number of enzyme in subsubclass 33

The Six Classes of Enzymes 1. Oxidoreductases Catalyze oxidation-reduction reactions - oxidases - peroxidases - dehydrogenases 34

2. Transferases Catalyze group transfer reactions 35

3. Hydrolases Catalyze hydrolysis reactions where water is the acceptor of the transferred group - esterases - peptidases - glycosidases 36

4. Lyases Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination 37

5. Isomerases Catalyze isomerization reactions 38

6. Ligases (synthetases) Catalyze ligation, or joining of two substrates Require chemical energy (e.g. ATP) 39

ENZYME KINETICS Why study enzyme kinetics? a) the precise scheduling of reactions in a cell is important to the cell and our understanding of its workings b) enzyme mechanisms, e.g., the number of kinetic steps and the detailed chemistry can be learned (enzymology). c) understanding enzyme function leads to better drugs.

Definition: rate of a reaction For an enzyme acting on its substrate, just as an ordinary chemical reaction, the rate of the reaction depends on the concentration of substrate, S. A reaction leading to formation of product is written: S P Rate = change in P /change in time or rate = v = Δ[P]/Δt

For a chemical reaction (as contrasted to an enzymatically catalyzed one), the rate is proportional to reactant [S]. A rate constant, k, can be defined: rate = v = Δ[P]/Δt = k [S] rate [S]

- In contrast, found empirically for enzymes: rate depends on [S] but hyperbolic curve & plateaus rate also depends on the enzyme concentration rate [S]

or Interpretation Michaelis-Menten Model E + S  E●S  E + P where E●S is enzyme-substrate complex, i.e., an intermediate complex. rate stops increasing or plateaus because the complex E●S becomes filled at high [S]

Assigning rate constants to MM model: k2 k3 E + S  E●S  E+ P k1 From this kinetic scheme, a relationship can be derived for the rate or velocity of the reaction: Michaelis-Menten Equation Vmax[S] [S] + Km gives hyperbolic curve on next slide. V =

Vmax Vmax is approached asymptotically Vmax/2 v or rate Km [S] Vmax, the maximum rate (plateau) is k3 x [total enzyme] Km =(k2 +k3)/k1, almost a binding constant Vmax Vmax is approached asymptotically Vmax/2 v or rate Km [S] Unit of velocity is μmoles/min×mg protein

Km = [S], where the velocity v = Vmax /2, is called the Michaelis constant. Km is in units of concentration Km good estimate for the optimum concentration of substrate. Vmax Vmax/2 v Km [S]

A plot of v = Vmax[S] [S] + Km is not accurate enough to derive good Km & Vmax. Computer analysis is done. Reciprocal Plot A double reciprocal plot or Lineweaver-Burk plot is linear and more eye-appealing for presentation. mathematically = “linear transformation”

Result is: 1/v = Km/Vmax●1/[S] + 1/Vmax Looks like the linear y = m●x + b m = slope b = intercept on y 1/v = Km/Vmax●1/[S] + 1/Vmax “x” Notice also intercept on “x” is -1/Km

1/v x x x x x x x slope = Km/Vmax intercept = 1/Vmax -1/Km 1/[S]

Competitive Inhibition presented as double reciprocal plot Model: E + S  E●S  E + P + I  E●I I resembles S I binds at active site reversibly E●I cannot bind to S so no reaction substrate inhibitor

Competitive Inhibition Vmax No I +I +more I Km In competitive inhibition, can always add enough [S] to overcome inhibition.  same Vmax

Competitive inhibition Double reciprocal plot + more I +I Same 1/v intercept, same Vmax Different slopes, competitive Inhibition changes apparent Km Note: inhibition line always above no inhibition. 1/Vmax No I 1/[S]

Molecular interpretation for competitive inhibition competitive inhibitor binds to same site as the substrate (competes). its structure usually resembles substrate or product. While the inhibitor is bound, the enzyme cannot bind substrate and no reaction possible. Many pharmaceutical agents are competitive inhibitors so are many toxic substances.

Blood pressure is regulated in kidney by renin, example: captopril Blood pressure is regulated in kidney by renin, a specific proteolytic enzyme, which acts on angiotensinogen, the precursor for the active regulator. renin angiotensinogen angiotensin I asp-arg-val-tyr-ile-his-pro-phe-his-leu converting enzyme angiotensin II the active factor O peptide captopril HS-CH2-CH-C-N COOH captopril is ACE inhibitor CH3 pro-like here (angiotensin converting enzyme}

Finding useful inhibitors Trial & error Molecular modeling Testing enzyme inhibition Testing safety Example: HIV Protease is a dimer. inhibitor is shown at active site. interactions involve both subunits.

Noncompetitive Inhibition E + S  E●S E + P + + I I   E●I  E●S●l substrate inhibitor E●I and E●S●I not productive, depletes E and E●S

Noncompetitive Inhibition + I 1/v No I 1/[S] different slopes, different 1/v intercepts.

Molecular Interpretation: Inhibitor binds the enzyme somewhere different from where the substrate binds. So the inhibitor does not care whether substrate is bound or not. Inhibitor changes the conformation of the enzyme at the active site so no reaction is possible with inhibitor bound. E●I and E●S●I not productive

Irreversible Inhibition reactive compounds combine covalently to enzyme so as to permanently inactivate it (previous examples are all reversible) almost all are very toxic most bind to a functional group in active site of enzyme to block that site

Example 1: diisopropyl fluorophosphate (DFP) binds covalently to serine in serine proteases & acetylcholinesterase - tool for biochemists sarin is a deadly nerve gas  Paralysis O Isopropyl-O-P-O-CH2- AChE CH3

Example 2: penicillin and related antibiotics bind covalently to a peptidase involved in cell wall synthesis in bacteria Staphylococci, Streptococci sp. and others