ENZYMES Classes based on type of reaction catalyzed Oxidoreductases catalyze oxidation-reduction reactions Transferases catalyze the transfer of functional

groups Hydrolases catalyze hydrolytic cleavages or their reversible reactions (split out H2O and add H+ and OH moieties to the substrate) Lyases catalyze the cleavage of bonds (C O, C C, C N bonds) without hydrolysis or oxidation-reduction reaction Isomerases catalyze isomerization reactions (rearrange functional groups) Ligases join two molecules; catalyze reactions involving the formation of bonds between two substrate molecules coupled to the cleavage of the pyrophosphate bond in ATP or another high energy compound Examples:

glucose +

O2
+

gluconic acid + H2O2

(oxidoreductase)
+

HOOC CH2 CH2 COOH + NAD

CHO H C OH HO C H H C OH H C OH CH2OH + ATP

HOOC CH CH COOH + NADH + H

(oxidoreductase)
CHO H C OH HO C H H C OH H C OH CH2 OPO3
2-

ADP

(transferase)

Sucrose

+ H2O

Glucose

Fructose
(hydrolase)

COOH H C OPO3 CH2OH

2-

COOH H C OPO3 CH2

2-

H2O

(lyase) L alanine D - alanine (isomerase)

Glucose
H3C C COOH + CO2 + ATP O

Fructose
(isomerase)
HOOC CH2 C COOH + O ADP + PO4 3

(ligase) ENZYMES Enzymes have enormous catalytic power. They catalyze reactions without affecting the equilibrium constant. They lower the activation energy of the reaction. The enzyme active site is the site which binds the substrate and where the substrate is converted into products. The site is a 3D entity which takes up a relatively small portion of the total volume of the enzyme. Specificity of binding depends on the precisely defined arrangement of the atoms in the active site.

In order to do its work, an enzyme must bind with at least one of the reactants. In most cases, the forces that hold the enzyme and its substrate are noncovalent (hydrogen bonds, ionic interactions, hydrophobic interactions) Most of these interactions are weak, so successful binding of enzyme and substrate requires that the two molecules be able to approach each other closely over a fairly broad surface. This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.

Characteristics of enzyme active sites Lock and Key Model only a substrate with the proper shape could fit into the enzyme active site

Induced fit Model

model assumes changes in active site structure as a substrate binds

Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.

Enzyme Kinetics
k

k+ 1 k-1

ES

E +

Assumptions 1. a first step in rapid equilibrium 2. second step is rate-limiting 3. conservation of enzyme Eo Km = [E][S] [ES] = = k[ES] E + ES

Total enzyme

Free ES enzyme complex

Eo = Km [ES] [S] ES = [Eo] Km + [S] [S]

ES

ES K m + 1 [S]

[Eo] [S] Km + [S]

= k [Eo] [S] Km + [S]

Vmax [S] Km + [S]

when enzyme is completely saturated with substrate, the reaction proceeds at its maximum possible rate

Lineweaver Burk linearization of the Michaelis Menten equation: 1/v = 1/Vmax + KM/ Vmax [S]

Plot of substrate concentration [S] vs velocity (v) plot

Lineweaver Burk

Factors Affecting Enzyme Action The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point.

Examples: the protease pepsin works best as a pH of 12 (found in the stomach) while the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine) Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve reflects the general effect of increasing temperature on the rate of chemical reactions. The descending portion of the curve above reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Enzyme Inhibition

Competitive reversible binds at active site substrate and inhibitor cannot bind the enzyme at the same time high substrate concentration outcompetes the inhibitor increases KM does not affect Vmax

Non-competitive reversible binds at site other than the active site substrate and inhibitor can bind the enzyme at the same time increased substrate concentration cannot outcompete the inhibitor has no effect on KM decreases Vmax

E + I

EI I E S

E I
ES E + P

ES I ESI

E + P

EI

Lineweaver Burk plots of inhibited enzymes

Effect of Inhibitors on the Michaelis-Menten rate equation, Apparent KM and Apparent Vmax Rate equation No inhibition Vmax [ S ] KM + [S]
Vmax [ S ] I 1 + K + [S] I

y-intercept
1 Vmax 1 Vmax

Apparent Vmax Vmax

slope
KM Vmax

Apparent KM KM

v =

Competitive inhibition

v= KM

Vmax

KM Vmax

[I ] 1 + [K ] I [I ] 1 + [K ] I

KM

[I] 1 + KI

Noncompetitive inhibition

v= KM

Vmax [ S ] 1 I Vmax + [S] 1+ K I

[I ] 1 +K I

1 + [I ] KI

Vmax

KM Vmax

KM

Holoenzyme -

conjugated enzyme

Apoenzyme - the protein portion of a holoenzyme - catalytically inactive when alone The non-protein portion may be: a prosthetic group (if covalently bonded to the protein) a coenzyme (if organic compound readily dissociable from the protein) a cofactor (if substance that takes part in enzymatic reactions but may be regenerated for further reaction e.g. metal ion) Metal ions may act as acid-base catalysts, form coordination compounds which aid in positioning of groups in a reaction for optimum catalysis. Some enzymes requiring metal ions Zn2+ alcohol dehydrogenase, carboxypeptidase 2+ Mg phophotransferase Fe2+, Fe3+ cytochromes, peroxidase, catalase Mn2+ arginase, pyruvate phosphokinase + 2+ Cu , Cu tyrosinase, cytochrome oxidase Some coenzymes and their reactions biotin carboxylation Acyl transfer Coenzyme A Flavin coenzymes Oxidation-reduction Lipoic acid Acyl transfer Nicotinamide adenine coenzymes Oxidation-reduction Tetrahydrofolic acid Transfer of one-C units Thiamine pyrophosphate Aldehyde transfer Pyridoxal phosphate transamination