Post on 03-Jan-2016
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 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 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 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 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 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 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 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 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 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 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 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