Prof. Dr. Azza Hassan AbouGhalia

1. Oxidoreductases :  Catalyze oxidation- reduction reactions. i.e. transfer of: Hydrogen atoms, Hydride ions, Electrons, or oxygen from one substrate to another. Prof. Dr. Azza Hassan AbouGhalia

2. Transferases: Catalyze transfer of a group (other than hydrogen) from a donor molecule to an acceptor molecule. e.g.  Amino transferase,  Acyl transferases,  Methyl transferase,  Phosphate group.  Glucosyl transferase. Prof. Dr. Azza Hassan AbouGhalia

3. Hydrolases: Catalyze cleavage of bonds ( of C-C, C-O, C-N, P-O, and certain other bonds by addition of water. e.g. Peptidases. Prof. Dr. Azza Hassan AbouGhalia

4. Lyases Catalyze Non-hydrolytic removal or addition of a group to a substrate. Example: Decarboxylase eliminates CO 2 Prof. Dr. Azza Hassan AbouGhalia

5. Isomerases: Catalyze geometric or structural changes within a single molecule. Examples:  Optical (D-L isomers) by racemase.  Geometric: Cis-Trans isomers.  Epimers.  Some mutases. Prof. Dr. Azza Hassan AbouGhalia

6. Ligases: Catalyze the joining (linking) together of two molecules, at the expense of high energy phosphate bond (from hydrolysis of ATP (or GTP). Examples:  Synthetases.  Carboxylases. Prof. Dr. Azza Hassan AbouGhalia

 Synthetases: belong to ligases.  Synthases: are a class of lyases & do not split ATP.

Study the effects of (change of) different factors on the rate of E catalyzed reaction

Variables (factors) that affect enzyme catalyzed reaction  Temperature,  pH,  Substrate concentration,  Enzyme concentration,  Coenzymes.  Metals

The initial velocity (vi)  Is measured when: a) very little substrate has reacted, or b) Very little product is formed.

The maximum velocity ( V max ) Is reached when:  All enzyme molecules are saturated with the substrate.

The rate (velocity ) of a reaction  The change (of the reactant to product)/ unit time.  Enzymes increase the rate of a chemical reaction. (Rate 1) (Rate 2)

The rate (velocity ) of a reaction Enzymes do NOT affect the:  Rate constant (k 1, K 2 ),  Equilibrium constant (K eq = K 1 /K 2 ),  The direction of flow of material.

 Over a limited range of temperature:  The velocity of an enzyme- catalyzed reaction increases as the temperature rises till it reaches a maximum at certain temperature (optimum temperature). Prof. Dr. Azza Hassan AbouGhalia

 37 ºC ( i.e. the body temperature) for many Enzymes in humans.  Close to the boiling point of water for microorganisms grow in natural hot springs.  N.B.: the changes in temperature is important in cold-blooded animals, but not in homeothermic animals. Prof. Dr. Azza Hassan AbouGhalia

 Between 0 & 40  C, most enzymes show a two fold increase in activity for every 10 °C rise in temperature.  i.e. Q 10 = 2.  The change in temperature is important in cold blooded animals & NOT in humans. Prof. Dr. Azza Hassan AbouGhalia

For most enzymes between 5 & 9 Pepsin: Acidic pH “2”. Salivary amylase: 6-8. Alkaline phosphatase? Lipp 2)

 In vivo: not important under normal conditions. The change in temperature.  In vitro: Important during measurement of enzyme activity in plasma & tissue samples. Prof. Dr. Azza Hassan AbouGhalia

New Tightly bound to the enzyme (prosthetic group) Loosely bound to the enzyme Has the same effect as the enzyme. Has the same effect of the substrate. Prof. Dr. Azza Hassan AbouGhalia 3)

Prof. Dr. Azza Hassan AbouGhalia  About 1/3 of all enzymes require metal ions for catalysis.  Metal ions help binding the substrate to the enzyme through formation of certain complexes: o Substrate bridge complex: E-S-M. o Enzyme bridge complex: M-E-S. o Metal bridge complex: S-M-E NewNew 4

Prof. Dr. Azza Hassan AbouGhalia 5)

Direct proportion. The velocity of a reaction is increased proportionally with the concentration of the enzyme ( if an excess substrate is present). If the [E] is halved, the (V 0 ), V max are reduced to half of the original. When the reaction velocity reaches equilibrium (maximum), the enzyme concentration has NO effect on the velocity of the reaction. Prof. Dr. Azza Hassan AbouGhalia

Rectangular hyperbola.

Prof.Dr.Azza Hassan AbouGhalia B KmKm Km Km

 Point B: half of the enzyme molecules are saturated with the substrate, the velocity is half maximal velocity (V max / 2).  The substrate concentration required to produce half the maximal velocity of the enzyme catalyzed reaction is termed Michaelis constant “K m ”

 First order reaction: --- When [s] <<Km, the velocity is proportional to the [s].  Zero order reaction: --- At high [S] the enzyme is saturated with respect to substrate. --- It occurs when [s] >>Km.., the velocity is constant & equal to V max ---i.e. the velocity is independent to the [S]. Prof. Dr. Azza Hassan AbouGhalia Zero order & first order reaction

Prof. Dr. Azza Hassan AbouGhalia 1) Definition: Is numerically equal to the substrate concentration at which the reaction velocity is equal to ½ V max.

Prof. Dr. Azza Hassan AbouGhalia 2) Constant for each enzyme with its substrate. 3) The K m value approximate the physiologic concentration of the substrate.

Prof. Dr. Azza Hassan AbouGhalia 4) Different organs can synthesize different variants of the same enzyme (isozymes) to act on the same substrate with different K m values. Example Glucose can be phosphorylated by: a) Glucokinase. b) Hexokinase.

Prof. Dr. Azza Hassan AbouGhalia 5) Reflects the affinity of the enzymes for its substrate [The lower the value of the K m, the higher is the affinity of the enzyme for its substrate].

Prof. Dr. Azza Hassan AbouGhalia Importance: Describes how the reaction velocity varies with substrate concentration. ○ 1)

Prof. Dr. Azza Hassan AbouGhalia 2) The Double- Reciprocal (Lineweaver –Burk) Plot  1/ V 0 = Y axis,  1/[S]= X axis,  the Y intercept=1/V max,  the negative intercept on the X axis-1/K m, so V max and K m can be calculated.

X-intercept Y- intercept

 Accurate calculations of K m & V max. from the linear plot.  Easier calculations of K m & V max. without the need of high substrate concentrations.  Helps identifying the mechanism of enzyme inhibitors (will be discussed).  Helps identify the sequence of binding of two substrates to one enzyme?. Prof. Dr. Azza Hassan AbouGhalia

 Is the amount of enzyme protein which converts 1µ mole substrate into product /per minute at 25 °C and optimal pH. (µ mole substrate/ minute). Prof. Dr. Azza Hassan AbouGhalia

 Is the number of enzyme units/ mg of protein (U/ mg protein). Prof. Dr. Azza Hassan AbouGhalia