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45 Cards in this Set
- Front
- Back
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requirement for life |
ability to effectively and selectively catalyze chemical reactions. biomolecules would be too slow to permit life without enzymes, |
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applications of catalyzing the reactions required for life |
many genetic disorders involve enzyme deficiency specific enzyme activites can serve as biomarkers drug molecules often influence specific enzymes commercial and industrial applications |
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requirements for full acitivty ezymes require: |
most enzymes only need the protein requirement but the others need:
cofactors: inorganic ions coezymes:organic molecules |
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prosthetic group |
a coenzyme or cofactor that is tightly associated with the enzyme. the difference is the degree of association
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six functional classes of enzymatic catalysis |
1. oxidoreductases: transfer of electrons (hydride ions or H atoms) 2. tramsferases: group transfer reactions 3. hydrolases: hydrolysis reactions (transfer of functional groups to water) 4. lyases: addition of groups to double bonds or formation of double bonds by removal of groups 5. isomerases: transfer of groups within molecules to yield isomeric forms 6. ligases: formation of C-C, C-S, C-O and C-N bonds by condensation reactions coupled to ATP cleavage |
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catalysts |
lower the amount of energy required for a reaction to proceed speed up attanment of equilibrium but do not change equilibrium are unchanged by the reaction (recycled to participate in another reaction |
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enzymes vs chemical catalysis |
faster: enzymes possess remarkable catalyic power, some approching catalyic perfection milder conditions: many chemical catalysts that require extremes of temperature, pressure and pH. enzymes function at physiological pH conditions
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how enzymes work |
enzymes catalyze the interconversion of sunstrate and product
E+S<----> ES<----->E+P
substrate (s): the molecule to be acted upon by the ezyme product(p): what is produced by the enzyme active site: the portion of the enzyme (E) responsible for binding the substrate leading to the formation of an ezyme substrate (ES) complex
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binding effects vs chemical effects |
BINDING EFFECTS 1) substrate binding 2) transition state stabilization
CHEMICAL EFFECTS 1) acid/base catalysis 2) covalent catalysis |
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substrate binding |
enzymes properly gather and position substrates. this makes the formation of the transition state more frequent= lowers the energy of activation
it promotes reactions by: reducing the entropy (decreased freedom of motion of two molecules in solution) desolvation of the substrate to expose reactive groups alignment of reactive functional groups of the enzyme with the substrate distortion of substrates induced fit of the enzyme in response to substrate binding |
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transition state stabilization |
an increased interaction of the enzyme and substrate occurs in the transition state (E-S) the enzyme distorts the substrate, forcing it toward the transition state an ezyme must be complementary to the transition state in shape and chemical character
active site must be similar enough to substrate to ensure specificity but different enough to promote change |
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transition state analogs |
are stable compounds whose structures resemble unstable transition states these analogs may be bound to the enzyme with higher affinity than the natural substrate these molecules may function as competitive inhibitors
catalytic antibodies may push the substrate towards the transition state to promote the reaction. similar to enzyme functinon |
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chemical modes of enzymatic catalysis |
after substrate binding, the enzyme can act upon the substrate to promote formation of the product. polar, ionizable residues participate. (ASP, GLU, HIS, CYS, TYR, LYS, ARG, SER)
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acid base catalysis |
reaction acceleration is achieved by catalytic transfer of a proton. side chains of some amino acids can act as a base (proton acceptors) or an acid (donors) histidine is often involved
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covalent catalysis |
substrate undergoes a state where it is based covalently to the enzyme to form a reactive intermediate two steps: first which forms a covalent linkage to the enzyme, second to regenerate the free enzyme
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enzyme kinetics |
kinetics is the study of the rates at which reactions occur. the rate of reaction quantified as the formation of product over time. enzyme kinetics measured in some unitt of concentration over time |
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variables that influence kinetics |
as enzymes are proteins, any variable that influences protein structures will influence enzyme activity rate will also be influenced by enzyme concentration and substrate concentration only interested in the relationship between initial velocity and substrate concentration |
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enzyme substrate complex |
enzymatic catalysis involves the formation of an enzyme substrate complex where the substrate is bound in the active site of the enzyme the substrate binds to the enzyme at the active site |
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velocity |
as velocity is defined as the change in product concentration over time it is necessary to measure product formation before equilibrium is reached |
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initial velocity |
velocity at the begining of an enzyme catatlyzed reaction prior to product accumulation |
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michaelis menton kinetics and the steady state assumptions |
had to assume that the rate of formation of the ES complex is equal to the rate of its breakdown
E+S=ES=E+P |
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michaelis menton steady state assumption |
rate of formation of the ES complex is equal to (E) (S) k1 rate of breakdown of the Es complex is equal to (ES)k-1+(ES) k2 therefore: (E)(S)k1=(ES)k-1+(ES)k2
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michaelis menton equation and graph |
describes the relationship between substrate concentration and initial velocity
vo= vmax(S)/km+(S)
vmax=maximum velocity of the enzyme km is (S) required to reach 1/2 vmax |
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enzyme turnover number |
the number of molecules of substrate converted to product per unit time under saturating conditions is calculated by vmax/(E) t
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lineweaver-burk plots |
also describe the relationship between (S) + vo are a double reciprocal plot of 1/vov.s./(S) are a more precise method of analysis of kinetic data are used to determine vmax+ km
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reversible enzyme inhibiton |
any inhibitor is a compound that binds to an enzyme to interfere with its activity inhibitors can prevent either formation of ES or the breakdown to E+P reversible inhibitors bind to the enzyme by non covalent interactions competitive and uncompetitive inhibtions |
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competitive inhibition |
usually resemble the substrate inhibitor binds only to free enzyme (E) not (ES) substrate cannot bind with I is bound at the active site vmax is the same but apparent km is increased can wash out the effects of the inhibitor |
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uncompetitive inhibition |
bind only to ES vmax decreased by conversion of ES to ESI km is decreased lines on double-reciprocal plots are parallel induced fit is seen
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irreversible enzyme inhibition |
irreversible inhibitors form stable covalent bonds with the enzyme to inactivate the enzyme suicidal inactivators are initially unreactive but are coverted to a reactive species that inactivates the enzyme. also called biochemical trojan horses or mechanism-based inactivators inhibitors of this enzyme are often lethal (sarin)
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properties of serine proteases |
digestive enzymes, including trypsin, chymotrypsin and elastase that cleave peptide bonds in protein substrates members of this family share similar sequences and active site residues are synthesized and stored in the pancreas as inactive zymogens to prevent damage to cellular proteeins catalytic mechanism contains elements of covalent and acid base catalysis |
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substrate specificity of serine proteases |
members of the serine protease family have unqiue substrate specficities (at what sequences they will cut polypeptide chains) trypsin cleaves by LYS and ARG chymotryposin cleaves by PHE and TYR elastase cleaves by GLY and ALA
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serine protease catalytic triad |
serine protease have a conserved catalytic mechanism based on a catalytic triad of residues (Asp, His, Ser) His removes H from Ser hydroxyl to make it a strong nucelophile as well as activating a water molecule to help regenerate the free enzyme (acid-base catalysis) Asp stabilizes the positvely charged His to facilitate serine ionization ser acts as a nucleophile attacking the carbonyl group of the polypeptide substrate (covalent catalysis) |
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chymotrypsin mechanism step 1 |
when substrate binds the side chain of the residue adjacent to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack binding is determined by the noncovalent interactions between the enzymes active site and the substrate complimentary. differ in specificity |
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chymotrypsin mechanism step 2 |
interaction of the Ser 195 and His 57 generates a strongly nucleophilic alkoxide ion on ser 195. the ion attacks the peptide carbonyl group, forming a tetrahedral acyl-enzyme. this is accompaned by formation of a short lived negative charge of the carbonyl oxygen of the substrate, which is stablized by hydrogen bonding in the oxyanion hole. regeneration original enzyme |
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chymotrypsin mechanism step 3 |
in stability of the negative charge on the substrate carbonyl oxygen leads to collapse of the tetrahedral intermediate. reformation of a double bond with carbon displaces the bond between carbon and the amino group of the peptide linkage, breaking the peptide bond. the amino leaving group is protonated by His 57, facilitatingits displacement covalent catalysis. |
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chymotrypsin mechanism step 5 |
an incoming water molecule is deprotonated by general base catalysis, generating a strongly nucleophilic hydroxide ion. attack of hydroxide on the ester linkage of the acyl-enzyme generates a second tetrahedral intermediate, with oxygen in the oxyanion hole again taking a negative charge
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chymotrypsin mechanism step 6 |
collapse of the tetrahedral intermediate forms the second product, a carbohydrate anion and displaces er 195
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chymotrypsin mechanism step 7 |
diffusion of the second product from the active site regenerates free enzyme |
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regulation of enzyme activity |
can be regulated by controlling the amount of the enzyme that is present (long term) or by adjusting the activity of a constant quantity of the enzyme (short term)
1. regulation of enzyme availability - location, rates of synthesis and degradation 2. regulation of enzyme activity a)covalent modification -phosphorylation methylation glyosylation b) non-covalent modification- allosteric regulation
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general properties of allosteric ezymes |
have activities that regulated by interactions with metabolic intermediates. allosteric modulators bind non covalently to allosteric enzymes at sites distinct from active site. are usually quaternary structure often catalyze branch point reactions often slow, catalyzing the rate limiting step of the pathway do not obey michaelis menten kinetics, have sigmodial curves rapid transition bewteen the active (R) and the inactive (T) conformations substrates and activators may bind only to the R state while inhibitors may bind only to the T site
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activation of phosphofructokinase by ADP |
the activity of PFK1 is responsive to the concentration of the substrate as well as the allosteric activators and inhibitors even at constant levels of substrate, the activity of enzyme can be modulated through changes in levels of the allosteric modulators |
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regulation by convalent modification |
many enzymes are regulated through the covalent linkage of e modifying group to changes some aspect of the proteins behavior, such as acitvity. a number of diff types of covalent modifications have been characterized most common post translation is through phosphorylation these modifications are usually reversible with one enzyme catalyzing the addition of the group and another enzyme catalyzing its removal |
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futile cycling |
running both anabolic and catabolic reactions at the same time. no progress, alot of heat. useful to warm up (brown fat) produces heat internal heaters avoided by reciprocal regulation
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reciprocal regulation |
never see these two enzymes active at the same time. ie) building a house when somone else is trying to break it down
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phosphorylation |
phosphorylation activates the catabolic enzyme and inactivates the anabolic enzyme in response to hormones released in the fed state (insulin) both enzymes are unphosphorylated when unphosphorylated, the anabolic enzyme is active and the catabolic enzyme is inactive |
