Enzyme Functions and Regulations

1

Exer Biochem c2-enzyme 2

Enzymes as catalysts Enzymes are proteins that catalyze different

chemical reactions that constitute our metabolism Speed up chemical reactions by lowering their energy

barrier (energy of activation), so that the reaction can take place at low temperature (37C)

May increase reaction speed by millions of times Active site: AA residues that bind substrates and

perform catalysis Binding site, catalytic site usually clefts in 3-D structure with specific AA residues

to interact with substrate 受質

Exer Biochem c2-enzyme 3

酵素的作用與能量釋放

Exer Biochem c2-enzyme 4

酵素作用模型 lock and key

Exer Biochem c2-enzyme 5Rates of enzymatic reactions:Substrate concentration

Initial velocity: enzyme reaction rate has to be measured quickly before accumulation of products

Plot Initial velocity vs [S] Usually hyperbolic (but not all), Michaelis-Menten

kinetics Vmax: maximum velocity

Enzyme active sites are totally saturated Michaelis constant (Km): [S] at ½ Vmax

Affinity of enzyme for its substrate: ↑Km, ↓affinity [S] usually Km in cells, quicker response to changes in ≦

[S] calculated more easily by Lineweaver-Burk plot

Exer Biochem c2-enzyme 6

Exer Biochem c2-enzyme 7

Isozymes, isoenzymes Enzymes catalyze same

reaction in different tissues, but have different kinetic parameters Hexokinase I, II, III, IV Low-Km isozymes

function when [glucose] is low, especially in brain

High-Km isozymes in liver, high [glucose]

Exer Biochem c2-enzyme 8Rates of enzymatic reactions:enzyme concentration

↑[enzyme], ↑Vmax in proportion No effect on Km

↑certain enzyme concentrations after exercise training adaptation

Exer Biochem c2-enzyme 9Environmental effects on enzyme function

Optimal pH Changes in active site or changes other sites that affect active sites

Optimal temperature Temperature too high cause protein denaturation Loss of enzyme activity Warming up muscles prior to exercise increase

enzyme activities

Exer Biochem c2-enzyme 10

pH and temperature affect enzyme velocity

Exer Biochem c2-enzyme 11

Turnover number (kcat) Turnover number (kcat), catalytic constant

Maximum number of substrate converted to product per enzyme active site per unit of time (usually per sec)

How fast an enzyme can convert substrate to product, Maximum catalytic activity for enzyme

Very diversified among different enzymes, can reach 106-107

Usually use kcat/Km: how fast the enzyme can work under physiological conditions

Far less diversified

Exer Biochem c2-enzyme 12

Enzyme inhibition

Competitive inhibitors Resemble normal substrate Bind to active site, but can not be changed into

product Noncompetitive inhibitors

Does not resemble normal substrate Does not bind to active site When bind to enzyme, it interferes with enzyme

function

Exer Biochem c2-enzyme 13

Exer Biochem c2-enzyme 14

Enzyme cofactors Enzymes may need other reactive groups (not AA)

for their functions Cofactors May be metal ions, Mg, Zn, Mn… May be organic molecules: coenzymes (e.g. NAD, FAD) Apoenzyme (inactive) + cofactor = holoenzyme (active)

Prosthetic group: a cofactor tightly bound to the enzyme at all times (e.g. heme in hemoglobin)

Deficiency diseases associated with inadequate intake of specific vitamins may due to insufficient catalytic power of enzymes

Exer Biochem c2-enzyme 15

Exer Biochem c2-enzyme 16

Exer Biochem c2-enzyme 17

Protein transporters Transmembrane proteins, integral membrane

proteins Technically not enzymes, but function consistently with

the kinetics of enzymes (Vmax, Km) Recognize specific transport substances, move them in a

particular direction Specific transport proteins: translocase, porter, carrier,

transporter, channel Facilitated diffusion: from higher to lower

concentration Active transport: from lower to higher

concentration Require energy: ATP or other chemical gradients Creatine transporter in muscle cell

Exer Biochem c2-enzyme 18Active transport: creatine transporter in muscle cells

Exer Biochem c2-enzyme 19Oxidation and reductions: redox reactions

Oxidation: something lose electrons Reduction: something gain electrons

These 2 reactions are always connected Dehydrogenation: hydrogen leaves as

electrons Oxidation reactions

Coenzymes that accept hydrogen (electron) NAD+ NADH; FAD FADH2

Exer Biochem c2-enzyme 20

Exer Biochem c2-enzyme 21

Exer Biochem c2-enzyme 22

Regulation of enzyme activity Control biological functions through control of

enzyme activities Synthesis/degradation of enzyme is time- and energy-

consuming Allosteric enzymes (allo: other), usually have

subunits Enzymes with sites other than active site that effectors

can bind and modify enzyme activity: allosteric sites Usually in metabolic pathways where they can control

the rate of flux of the entire pathway (rate-limiting enzymes, e.g. phosphofructokinase)

Ligand: molecule that bind to large molecules Ligands can bind to allosteric sites and increase or

decrease enzyme activity

Exer Biochem c2-enzyme 23

Exer Biochem c2-enzyme 24

Regulation of enzyme activity

Ratio of positive/negative effectors (activator/inhibitor)

Feedback inhibition Enzyme activity inhibited by its product

Allosteric regulation is ‘fine-tuning’ type of enzyme activity modulation

Exer Biochem c2-enzyme 25

Exer Biochem c2-enzyme 26Regulation of enzyme activity: Phosphorylation, dephosphorylation

Covalent modification of enzymes Rapidly turn on or off enzyme activity

Phosphorylation, dephosphorylation Add/remove a phosphate group in specific AA Catalyzed by protein kinase, phosphoprotein

phosphatase Critical in controlling and integrating

metabolism Controlled by hormones, cytokines, other factors Signal transduction pathways

Exer Biochem c2-enzyme 27

Phosphorylation/dephosphorylation

Exer Biochem c2-enzyme 28Regulation of enzyme activity: Thiol oxidation and reduction

Thiol oxidation and reduction: redox control of enzyme functions Usually on cysteine thiols (-SH) reactive oxygen species (superoxide), reactive nitrogen

species (nitric oxide) When proteins are oxidized: thiols may form sulfenic (P-

SOH) , sulfinic (P-SOzH), or sulfonic (P-S03H) acids; intra- or interprotein disulfides (P-S-S-P); nitrosothiols (P-SNO), glutathione (P-S-SG)

can be reversed by specific protein-reducing enzymes called glutaredoxins, thioredoxins, peroxiredoxins

Exer Biochem c2-enzyme 29

irreversible

Exer Biochem c2-enzyme 30

Exer Biochem c2-enzyme 31Measurement of phosphorylated proteins

Western blot Serine, tyrosine, threonine Serine kinase, tyrosine kinase Antibody to phospho-serine, phospho-

tyrosine Or specific Phosphorylated protein Phosphorylated/total Total GS, phophorylated GS (203-Ser

phosphorylated GS, 872-Thr-phosphorylated GS)

Stripping buffer

Exer Biochem c2-enzyme 32

Measurement of enzyme activity Usually measure maximal activity

[S] high enough to generate true Vmax Standardized pH, temperature Simple method to measure [S] or [P], e.g. color

Use NADH disappearance/appearance Absorption at 340 nm One international

1 international unit (IU) of enzyme activity:the amount of enzyme that converts one micromole of substrate to product in one minute Usually IU/mg tissue, IU/ml