Chapter 6 Enzymes: The Catalysts of Life
many thermodynamically feasible reactions in a cell that could occur do not proceed at any appreciable rate
ex: hydrolysis of ATP has ΔG = -7.3 kcal/mol
ATP + H2O = ADP + Pi
however, ATP dissolved in water remains stable for several days
activation energy (EA) - minimum amount of energy equired before collisiion between the reactants will give rise to products
molecules that could react with one another often do not because they lack sufficient energy; each reaction has a specific EA
transition state - reactants need to reach an intermediate chemical stage
has higher free energy than that of the initial reactants
the rate of a reaction is always proportional to the fraction of molecules with an energy equal to or greater than EA
the only molecules that are able to react at a given time are those with enough energy to exceed the activation energy barrier
most rxn at norm cell temp, the EX is so high that few molecules can exceed the EA barrier
reactions that are thermodynamically unstable but lack sufficient EA are said to be in metastable state
life depends on high EA that prevent most reactions in the absense of catalysts
EA barrier must overcome in order for needed reactions to occur; either increasing the energy content of molecules or lowering the EA requirement
input of heat can increase the KE of the avg mole, ensuring that more moles will be able to take part in a reaction- not useful incells because they are isothermal
catalyst - enhances the rate of a reaction by providing a surface for efffective interaction between reactant molcules; lowering EA
proceed throught the reaction unaltered
3 basic properties
increase reaction rates by lowering the EA required
form transient, reversible complexes with substrate molecules
change the rate at which equilibrium is achieved, not the position of the equilibrium
organic catalysts are enzymes
most enzymes are known to be proteins. some RNAs have enzyme activites called ribozymes (aqualysis - a protease, hepatitis delta virus ribozyme)
every enzyme contains a characteristic cluster of amino acids that forms the active site, where substrates bind and catalysis takes place
usually a groove or pocket that accommodates the intended substrates with high affinity
amino acids found in active cites mainly are charged or polar: histidine, serine, aspartate, glutamate, lysine
cofactors
some enzymes contain nonprotein cofactors needed for catalytic activity often because they function as electron acceptors
these are called prosthetic groups and usually metal ions
located at the active site and are indispensable for enzyme activity
small organic molecules called coenzymes
requirements for trace amounts of vitamins and minerals for functions of many enzymes
enzyme specificity:
Because of the shape and chemistry of the active site, enzymes have a very high substrate specificity
inorganic catalysts are very nonspecific, wheras similar reactions in biological systems generally have a much higher level of specificity
group specificity: carboxypeptidase A
high specificity: succinate dehydrogenase
enzyme diversity and nonmenclature
thousands of different enzymes have been identified with enormous diversity
names have been given to enzymes based on substrate (protease, ribonuclease, amylase) or function (methyl-transferase)
under the enzyme commision (EC) - enzymes are divided into 6 major classes based on general function
class - reaction type - ex - reaction catalyzed
oxidoreductases - oxidation:reduction reactions (electron transfer) - alcohol dehydrogenase - oxidation of ethanol to acetaldehyde as NAD+ is reduced to NADH
transferases - transfer of functional groups from one molecule to another - hexokinase - phosphorylation of glucose to glucose-6-phosphate using the terminal phosphoryl group from ATP
hydrolases - hydrolytic cleavage of one molecule into two molecules - glucose-6-phosphatase - cleavage of glucose-6-phosphate into glucose and inorganic phosphate using a water molecules
lyases - removal of a group from or addition of a group to a molecule - pyruvate decarboxylase - removal of a carboxyl group from pyruvate to produce acetaldehyde and CO2
isomerases - movement of a functional group within a molecule - maleate isomerase - a cistrans isomerization of maleate to fumarate
ligases - joining of 2 molcules to form a single molecule - pyruate carboxylase - additon of CO2 to pyruvate to rpoduce oxaloacetate
Enzymes are characterized by sensitivity to temperature
not a concern in homeotherms (birds and mammals) which maintain a constant boyd temperature
but many organisms (poikilotherms) function at their environmental temperature, can vary widely
optimal temperature
temperature range over which an enzyme denatures varies amond enzymes and organisms
reaction rate of human enzymes is max at 37C (optimal temp) the normal body temperature
most enzymes of homeotherms are inactivated by temperatures about 50-55C
sensitivity to pH
most enzymes are active within a pH range about 3-4 units
pH dependence is usually due to the presence of charged amino acids at the active site or on the substrate
pH changes affect the charge of such residues and can disrupt ion and hydrogen bonds
sensitivity to other factors
enzymes are sensitive to things such as molecules and ions that act as inhibitiors/activators
most enzymes are also sensitive to ionic strength of the enivronment
affects H bonding and ionic interactions neeed to maintain tertiary conformation
substrate binding, activation and catalysis occur at the active site
bc of the precise chemical fit between the active site of the enzyme and its substrates, enzymes are highly specific
once in the active site, substrate molecules are bound to the enzyme surface in the right orientation to facilitate the reaction
substrate binding usually involves hydrogen bonds or ionic bonds or both
substrate binding is readily reversible
Emil Fischer (1894) - proposed that the enzyme ws seen as ridig with the substrate fitting into the active site like a key in a lock (lock and key model)
induced-fit model (daniel koshland 1958) - substrate binding at the active site induces conformational changes in the shape of both the enzyme and substrate
conformational change
induced conformational change brings needed amino acid side chains into the active site even those that are not nearby
once in the active site, the substrate is held in place by specific noncovalent interactions
theres position the substrate optimally for catalysis and distinguish the real substrate from similar molecules
substrate activation - role of the active site to recognize and bind the appropriate substrate also to activate it by providing the right envirionment for catalysis; several possible mechianisms
bond distortion = makes the bond more susceptible to catalytic attack
proton transfer = increases reactivity to substrate
electron transfer = results in temporrary covalent bonds b/w enzyme and substrate
sequence of catalytic events
random collision of a substrate molecule with the active site results in its binding there
substrate binding induces a conformational change that tightens the fit, facilitating the conversion of substrate into products
the products are then released from the active site
the enzyme molecule returns to the original conformation and the active site is now available for another molecule of substrate
ribozymes - catalytic RNA molecules (1980s)
the earliest enzymes were likely catalytic self-replicating RNA moles
thomas cech 1981 discovered rna was self-splicing
ribnuclease P = enzyme that cleaves transfer RNA precursors to yield functional RNA moles - has RNA and protein componenet
sidney altman showed that only the RNA componenet was capable of performing the cleavage
hairpin ribozyme - RNA region that catalyzes RNA processing reactions essential for replication of satellite RNA molecules in which it is embedded
ribosomal RNA = active site for peptidyl transferase activity on the large ribosomal subunit is one of the rRNA mole (Hally Noller 1992)
enzyme kinetics = describes the quantitative saspects of enzyme catalysis and the rate of substrate conversion into products
reaction rates are influnced by factors like concentration of substrates, products and inhibitiors
can help understand the nature of enzyme activities in human diseases to assist the design of drugs that can inhibit the enzyme activites
help parmaceutic companies to optimize their manufacturing processes, maximixing protein production for health related applications and research
help developing enzyme based assays dor diagnostic applications
monkeys “enzyes” shelling peanuts “substrates” - 10 monkeys equally good at finding and shelling peanuts that are equally distributed on the floor
if more peanuts were added, at some point the monkeys would have easy access to the peanuts that adding even more yet would not allow them to work any faster (E+S → ES) and we’d be at the saturation point = V max
initial reaction velocity (v) = rate of change in product concentration per unit time, depends on the substrate concentration [S]
at low [S], doubling [S] will double
as [S] increases, each additional increase in [S] results in a smaller increase in v
v= Vmax[S] / Km+[S] michaelis-menton equation
Km = Michaelis constant = concentration of substrate that gives half-max velocity
very low substracte concentration ([S] « Km)
v = Vmax[S] / Km
v is proportional to [S], v of an enzyme catalyzed reaction increases linearly w [S]
very high substrate concentration ([S] » Km)
v = Vmax
initial velocity of the reaction is independent of the variation in [S], and Vmx is the velocity at saturating substrate concentrations
only way to increase Vmax is to increase enzyme concentration
([S] = Km)
v = Vmax/2
Km is the specific substrate concentration at which the reaction proceeds at ½ its mac velocity
the lower the Km value for a reaction, the lower the [S] range in which the enzyme is effective
Vmax is important as a measure of the potential maximum rate of the reaction
knowing Vmax and Km and the in vivo substrate concentration helps estimate the likely rate of the reaction under cellular conditons
kcat = Vmax / [Et]
turnover number - rate at which substrate molecules are converted to product by a single enzyme at maximum velocity
Km and kcat vary greatly among enzymes
lineweaver-burk double reciprocal equation: 1/v = Km/Vmax (1/[S]) + 1/Vmax
enzyme inhibitors
enzymes are influcenced (mostly inhibited) by products, alternative substrates, substrate analogues, drugs, toxins, and allosteric effectors
the inhibition of enzyme activity plays role as control mechanism in cells
drugs and poisons frequently exert their effects by inhibition of specific enzymes
inhibitor
irreversible inhibitor - binds enzyme covalently, cause permanent loss of catalytic activity and are gnerally toxic to cells (binds tightly to enzyme)
cyanides, heavy metals , nerve gas poision, some insecticles
aspirin binds and irreversibly inactivate cyclooxygenase-1 (COX-1)
COX-1 functions: prudction of prostaglandings, other signal molecules - inflammation, constriction of blood vessels and platelet aggregation
reversible inhibitor - bind enzymes noncovalently and can dissociate from the enzyme
competitive inhibitor - inhibitor and substrate both bind to the active site of the enzyme. binding of an inhibitor prevents substrate binding → inhibiting enzyme activity
malonate, ethanol (binds to active site)
noncompetitive inhibitor - inhibitor and substrate bind to different sites on the enzyme. binding of an inhibitor distorts the enzyme. binding of an inhibitor distorts the enzyme, inhibiting substrate binding/reducing catalytic activity
isoleucine; feedback inhibitor (binds at toher than active site)
enzyme regulation
enzyme rates must be continously adjusted to keep them tuned to the needs of the cell
substrate-level regulation = regulation that depends on interactions of substrates and products with an enzyme
increases in substrate levels result in increased reaction rates, whereas increased product levels lead to lower rates
feedback (endproduct) inhibition - final product of an enzyme pathway negatively regulates an eariler step in the pathway
usually enzymes regulated this way catalyze the 1st step of a multistep sequence
by regulating the 1st step of process, cells can regulate the entire process
allosteric regulation
allosteric enzyme: regulated by moles other than reactants and products
single most important control mechanism whereby the rates of enzymatic reactions are adjucted to meet the cells needs
are large multisubunit proteins with active/allosteric site on each subunit
active site = catalytic subunit
allosteric site = regulatory subunit
bindinf of allosteric effectors alters the shape of both catalytic and regulat subunits
allosteric inhibition - enzyme subject to allosteric inhibition is active in the uncomplexed form which has a high affinity for its substrate (S). binding of an allosteric inhibitor stablizes the enzyme in its low affinity form → results in little/no activity
allosteric activation - enzyme subject to allosteric activation is inactive in its uncomplexed form, has low affinity for its substrate. binding of an allosteric activator stablizes the enzyme in its high affinity form → results in enzyme activity
many allosteric enzymes exhibit cooperativity - as multiple catalytic sites bind substrate molecules, the enzyme changes conformation whcih alters affinity for the substrate
positive cooperativity - conformation change increases affinity for substrate
negative cooperativity - affinity for substrate is decreased
covalent modification - active is regulated by addition or removal of groups such as phosphate, methyl, acetyl groups (reverisble additon of phoshpate)
phosphorylation - occurs most commonly by transfer of a phosphate group from ATP to the hydroxyl group of serine, threonine, or tyrosine residues in a protein
dependding on enzyme, may be assoicated with activation/inhibition of hte enzyme
protein kinases - catalyze the phosphorylation of other proteins
dephosphorylation - removal of phosphate groups from proteins, is catalyzed by protein phosphatases
proteolytic cleavage = activation of a protein by a 1-time, irreversible removal of part of the polypeptide chain
proteolytic enzymes of the pancreases (trypsin, chymotrypsin, carboxypeptidase - ex of enzymes synthesized in inactive form (as zymogens) and activated by cleavage as needed)