Enzymes Flashcards
enzymes definition
protein catalysts (usually) that increase the rate of reactions without being changed in the overall process
Roles of enzymes in human biochemistry
convert substrates into products and channel them into useful pathways, and so direct all metabolic events
How do enzymes
increase the rate at which reactions occur, but do not invent new reactions
is life possible without enzymes?
no
Nomenclature
-ase at end of substrate name or description of the action performed (lactate dehydrogenase)
Some enzymes retain original trivial name.
What are the 6 major classes of enzymes
- oxidoreductases
- transferases
- hydrolases
- lyases
- isomerases
- ligases
oxidoreductases
catalyze oxidation-reduction reactions, such as
transferases
catalyze transfer of C-, N-, P- containing groups, such as
Hydrolases
catalyze cleavage of bonds by addition of water, such as
Lyases
catalyze cleavage of C-C, C-S and certain C-N bonds. opposite function as ligases
isomerases
catalyze racemization of optical or geometric isomers
Ligases
Catalyze formation of bonds between carbon and O, S, N coupled to hydrolysis of high energy phosphates. opposite function of lyases
systemic name
divided into the 6 major classes of enzymes, includes the names of all the substrates in the reaction catalyzed + ase. Each enzyme is also assigned a unique number
Synthase
No ATP required
Synthetase
requires ATP
Phosphatase
Uses H2O to remove phosphoryl group
Phosphorylase
uses Pi to break a bond and generate phosphorylated product
oxidase
uses O2 as acceptor without incorporating it into a reaction
Oxygenase
one or both oxygen atoms are incorporated
Dehydrogenase
electron acceptor in a redox reaction NAD+/FAD+
Arrows used in reactions
indicate the direction of the reaction under the normal physiological range of conditions
If the arrow goes in both directions
can catalyze either reaction
if the arrow goes only in one direction
it will only catalyze the reaction in the direction indicated by the arrow. Does not mean the opposite reaction cannot occur, just not under that enzyme
Enzyme structure
active site, allosteric site
catalytic properties of enzymes
efficiency, specificity
some other enzyme properties
- requirements for non-protein molecule
- regulation
- subcellular localization
active site
- special ‘pocket’ or ‘cleft’ that binds the substrate (3D shape)
- formed by folding of the protein molecule that allows specific amino acid side chains to participate in S binding and catalysis
- Binding of S produces ES complex which induces conformational change in the enzyme that facilitates catalysis (induced model fit)
- ES is converted into an EP complex, this complex dissociates to enzyme and product
allosteric site
any other part of the enzyme molecule that is different from the active site. Binds different regulatory molecules
Efficiency
- 10^3-10^14 faster than uncatalyzed reactions
- turnover number (Kcat) is the number of substrate molecules converted to product per enzyme molecule per second
specificity
- highly specific
- only one or a few substrates
- only one type of chemical reaction
- the set of enzymes present in a cell determines which reactions will occur in that cell
coenzymes
organic molecules that are required by certain enzymes to carry out catalysis
vitamin derivatives
NAD+, FAD, NADP+, CoQ, CoA
Cofactors
inorganic substances that are required for, or increase the rate of, catalysis
Usually ions
Zn2+, Mg2+, Fe3+, Fe2+
holoenzyme
enzyme + nonprotein component = active
apoenzyme
enzyme without nonprotein component=inactive
regulation by inhibitors and activators
a negative feedback mechanism, the amount of the end product produced is regulated by its own concentration
post translational modifications
regulation through covalent modulation of the enzyme molecule such as phosphorlyation, myristoylation, and glycosylation
enzyme protein production
transcription and translation of enzyme genes
regulation through specific local environment
high pH or temp to low pH or temp
enzyme compartmentalization
different metabolic pathways occurring in different cellular compartments
Advantages of the enzyme compartmentalization
isolates substrates and products from competing reactions
How enzymes work
- energy changes occurring during the reaction
2. chemistry of the active site
Free Energy (Gibbs free energy, G)
quantitative measure of the energy transfers between chemical reactions
energy barrier
energy difference between that of reactants (A) and high energy intermediate (T*) that occurs during formation of a product (B)
Free energy of activation
the difference in free energy between the reactants and T*
How do enzymes work
lowering the free energy of activation without affecting the energies of the reactants and products
chemistry of active site
active site is a dynamic molecular machinery.
the enzyme is returned to its unaltered state before the release of the product
stabilization of the transition state
- creates a microenvironment in which the bonds in the intermediates are not like the ones in the substrates and products
- this greatly increases the concentration of the reactive intermediates that can be converted to products
provide chemical groups that
- participate in reactions with the substrate
- enhance the probability of transition state formation
factors affecting reaction velocity
- substrate concentration
- temperature
- pH
rate of velocity (v)
number of substrate molecules converted to product per unit time (micromole of product formed per minute)
Maximal velocity (Vmax)
the rate of an enzyme catalyzed reaction increases with substrate concentration until Vmax is reached.
it represents the saturation of all available binding sites on enzyme molecules in the reaction
hyperbolic curve
most enzymes show michaelis-menten kinetic when initial reaction velocity (Vo) is plotted against substrate concentration ([S]), which results in a hyperbolic curve.
sigmoidal curve
some enzymes that are allosterically regulated or demonstrate cooperativity
Increase of V with temp
reaction velocity increases with increasing temp until a peak is reached. An increase in temp would increase the number of molecules that have the energy to overcome the energy of activation
decrease of V with higher temperature
further increase in temp beyond the peak velocity results in a decrease in velocity usually as a result of denaturation of the enzyme
what is the temp optimum for humans
37 C
what temp does denaturation of enzymes being in humans?
40 C, why high fevers are potentially life threatening
Effect of pH on the ionization of the active site
E and S usually have to have certain groups in a protonated or un-protonated form to interact optimally for catalytic activity. Any change in pH may influence this state resulting in decreased reaction rate
effect of pH on enzyme integrity
protein structure itself require specific ionic interactions that are influenced by the pH, hence, higher or lower pH can denature the enzyme
variable pH optimum
the pH optimum is specific characteristic of each enzyme and often reflects the pH of the environment in which the enzyme functions in the body.
digestive enzyme
reaction model for michaelis-menten
- a simple model that accounts for most of the features of enzyme-catalyzed reactions
- E reversibly combines with its S to form an ES complex that subsequently yields the P and regenerates the free E
Michaelis-menten equation
describes how reaction reaction velocity varies with substrate concentration at a given concentration of enzyme
vo=(Vmax[S])/(Km + [S])
Assumptions about the MM equation
- the concentration of S is much greater than the concentration of E
- [ES] does not change with time
- the initial velocity (vo) is used in the analyses
Km
- characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that particular substrate
- defined as the amount of substrate needed to half maximal velocity (1/2 Vmax)
small Km
high affinity, as it takes a low concentration of substrate to reach 1/2Vmax
high Km
low affinity, as it takes a high cxn of substrate to reach 1/2 Vmax
relationship of velocity to [E]
reaction rate is directly proprtional to [E] at all cxns of [S]
When [S] is much lower than Km
velocity is appx proportional to the substrate cxn, and the rate is said to be first order with respect to the substrate
when [S] is much greater than Km
the velocity is appx constant and equal to Vmax, and independent od [S], and the rate is said to be zero order with respect to the substrate
lineweaver-burk plot
- uses 1/vo vs 1/[S], which results in a straight line, which allows us to determine both the Km and the Vmax
- intercept on x-axis is equal to -1/km
- the intercept on the Y-axis is equal to 1/Vmax
- very useful for determining the mechanism of action of enzyme inhibitors
If we increase Km or Vmax on Lineweaver Burk plot…
they will be closer to zero
if we decrease km or Vmax on a lineweaver burk plot…
they will be further from zero
inhibitors
any substance that can diminish the velocity of an enzyme-catalyzed reaction
Irreversible inhibitor
bind to E through cavalent bonds anf the only eay to recover E-activity is to synthesizea new molecule
suicide inhibitor-E
converts I into a reactive form in its active site
reversible inhibitors
bind through non-caovalent bonds, which allow for recovery of E-activity
competitive reversible inhibitor
I competes with S for the active site of the E
noncompetitive reversible inhibitors
I binds to a different (allosteric) site of the E molecule
Binds reversibly to a different site on the enzyme than the substrate. It may bind to either the free enzyme of the ES complex
Competetive inhibition on Vmax
the effect of a competitive I can be overcome by high [S], therefore the same Vmax can be obtained, no effect on Vmax
competitive inhibition effect on Km
a competitive I increases the apparent Km for a given substrate and more substrate is needed to achieve 1/2 Vmax, therefore the apparent affinity is lower. Km is increased
HMG CoA-reductase
the rate-limiting enzyme in cholesterol biosynthesis
statin drugs are
structural analogs of HMG-CoA reductase substrate that: compete for the active site of the enzyme. By inhibiting de novo synthesis of cholesterol, they help lower plasma cholesterol levels
noncompetitive inhibition effect on Vmax
effect cannot be overcome by increasing [S], therefore they lower the apparent Vmax of the reaction. Vmax is decreased
noncompetitive inhibition effect on Km
do not interfere with binding of the substrate to the enzyme (affinity). No effect on Km
Noncompetitive inhibition of purine degradation
noncompetitive inhibitor which reduces the production of uric acid, used for treatment of hyperuricemia and related medical conditions
what regulates enzymes
effectors (modifiers) that bind to the sites other than the active site not he enzyme non-cavalently
What can effectors do?
- alter the affinity of the enzyme for its substrate (affect Km)
- alter the maximal catalytic activity (affect Vmax)
- alter both
- be negative
- be positive
How does an effector altering the affinity of the enzyme for its substrate have an affect?
it has an affect on Km
What does an effector altering the maximal catalytic activity do?
affect Vmax
negative effector
inhibit enzyme activity
decrease Vmax and/or increase Km
positive effector
increase enzyme activity
increase Vmax and/or decrease Km
Homotropic effector
substrate itself serves as an effector
usually +, so enhances the catalytic properties of the active site, also called “cooperativity effect
What kind of curve does cooperatively show?
sigmoidal, does not follow MM kinetics
What kind of curve would you see for a homotropic effector?
sigmoidal
heterotropic effectors
effector is different from the substrate molecule
feed-back inhibition
feed back inhibition
an end-product of a metabolic pathway inhibits upstream step
phosphofructokinase-1
rate limiting enzyme in glycolysis
allosterically inhibited by high levels of substrate
this inhibition redirects glucose towards the synthesis of glycogen
What is the most commonly encountered type of enzyme regulation by covalent modification?
phosphorylation/dephosphorylation
what AA can be phos/dephos?
serine, threonine, tyrosine
What kind of enzymes phos/dephos
protein kinases - phosphorylate, use ATP as phosphate donor
Protein phosphatases-dephosphorylate or remove a phosphate group
the amount of enzyme present can be controlled by altering the rate of
synthesis (altering gene expression-increase/decrease)
degradation (targeting for proteolysis
timing: gene expression regulation
takes from hours to days to see an effect on reaction rates (effects of steroid drugs are not immediate)
timing: covalent changes
immediate to minutes
timing: allosteric changes
immediate
plasma
physiological fluid part of the blood without the cells (contains coagulating factors)
serum
plasma without the coagulating facts (prepared from blood in laboratory)
actively secreted plasma enzymes
have certain function in plasma
small group
do not play role in diagnosis
plasma enzymes not actively secreted
function intracellularly not in plasma released from cell lysis during normal turnover fairly constant increase=tissue damage
alanine aminotransferase
released from damaged liver cells, it is part of the liver function
