Enzymology

Pre-steady state

- analyzed at high [E] - need specialized equipment to obtain data - obtain microscopic rate constants - can obtain detailed mechanistic information - intermediates can be directly observed

Steady state

- analyzed at low [E] - easy to obtain data - obtain macroscopic rate constants - few mechanistic details - coupled with other methods to obtain mechanistic details

Enzyme functions

- cell shape and motility - surface receptors - metabolism - cell cycling - protein synthesis - transcription - hormone release - muscle contraction

Why study?

- determine if biological feasible rxn in body - determine if intermediate structure is correct for analog development - determine the progression of mechanism - determine if intermediate or product forms first - can see if intermediate formation is faster than breakdown - can target intermediate if it lasts a long time

Applications of TS theory & Hammond

- determining structure of TS can lead to design of TS analogs with similar ground-state geometry to TS - analogs can be used as enzyme inhibitors in drug therapy - analogs can be used to determine mechanism of enzyme-mediates rxns

Burst kinetics

- stoichiometric release of one of products is much faster than SS Vmax - intermediate formation is faster than its breakdown, can see a burst of product formation after intermediate formation - PNP is released in PSS burst in mole:mole amount with concentration of active sites present - rate slows to Vmax - initial phase of rapid acylation of chymotrypsin, then slow hydrolysis of acyl-enzyme intermediate (rate-determining)

PSS study requirements

- time range capability of seconds-nanoseconds to capture the Kcat range of most enzymes - enzyme concentrations in substrate quantities in order to see the enzyme, rely on contributions from recombinant DNA for abundant enzyme amounts - techniques capable of detecting short-lived species

2 syringe stop-flow

- two syringes are filled with enzyme or substrate solution and compressed into mixer 1 - another syringe uses air to force the mixture into mixer 2 to react with a 3rd reactant from another syringe - final mixture is compressed into observation cell and flow is stopped This can be used to assess multiple factors, such as inhibitors, activators, etc.

Pre-steady state studies

- visualize how many ES intermediates occur during a single turnover - determine order of rxn, including rate-determining step - study effects of point mutations on intermediate formation - determine enzyme folding mechanism during rxn - trap and characterize makeup of each ES intermediate Km and Kcat can give idea of time needed to complete a single turnover (Kcat) and how much E or S needed for PSS determinations (Km).

Catalyst

1. Chemical or biological substance or structure that lowers activation energy 2. Accelerates the rate of a chemical rxn

Steady state conditions

1. Enzyme is fully saturated with substrate 2. Rxn rates change slowly in response to changing [S] 3. Easier to obtain rates experimentally 4. Concentration does not change over time since the rate of formation and decomposition of ES complex will be constant k1[E][S] = k-1[ES] + k2[ES] rate of formation = rate of decomposition

Pre-steady state conditions

1. Enzyme is not saturated 2. [ES] increases with [S] 3. Can calculate fastest steps in only first rxn turnover

How to alter pKa's

1. Ionizable groups buried in a hydrophobic environment - causes groups to take on neutral form * pKa of negative groups (Asp, Glu, Cys) increases * pKa of positive groups (Lys, Arg) decreases - can pair Asp with Glu residue or bulky hydrophobic residue - can pair Glu with Lys and Arg to drive up pKa 2. Ionizable groups exposed to electrostatic environment - for opposite charge: * pKa of negative groups decrease * pKa of positive groups increase - for like charge: * pKa of negative groups increase * pKa of positive groups decrease - can place 2 Glu in close proximity: one has pKa driven up to become protonated (neutral), and one remains deprotonated

How to determine e

1. Measure the intensity of absorbed light for solutions of various concentration 2. Construct a plot of A vs. c, which should be a straight line 3. Plot the line of best fit through the points, the slope of this line is e for a particular path length

Detection of actual intermediates

1. Must be isolated and characterized 2. Must be formed sufficiently rapidly to be on rxn pathway 3. Must react sufficiently rapidly to be on rxn pathway Requires PSS measurements for intermediate formation and decomposition rates. Requires SS measurements to compare intermediate rate constants with overall catalytic activity of enzyme rxn. Intermediates are distinct from the transition state. They have bonds and are fully formed. They can be found in the rxn using stop-flow or quench.

Nucleophile groups in enzymes

1. OH - in serine, forms an acyl or phosphoryl enzyme int 2. SH - in cysteine, forms an acyl or phosphoryl int 3. COO- - in aspartic/glutamic acid, forms acyl enzyme int 4. NH2 - in lysine, forms a Schiff base 5. imidazole - in histidine, forms an acyl enzyme int 6. OH - in tyrosine, forms a phosphoryl enzyme int Many of these groups also make good acid-base catalysts because they contain unshared electron pairs that can be used in donation, or can abstract protons.

Enzyme properties

1. Vital for chemical rxns in the cell through breaking, forming, and rearranging bonds on a substrate 2. Modify substrate (now a product) to perform a different task 3. Able to: - transform energy and matter in the cell - perform cell-cell and intracellular communication - allow for cellular homeostasis to persist

Denaturation of enzymes

1. pH change or temp change - can be controlled in lab setting 2. AA sidechains altered by oxidation - usually with cysteines, lysines - lose catalytic integrity 3. Enzyme can cleave itself - ex. proteases in trans conformation will cleave themselves and become inactive

Beer's Law

Absorbance displays a simple dependence on the concentration of the analyte and cell path length. A = ecl e - molar absorptivity c - concentration l - length of sample light passes through, in cm

Catalytic base

Abstracts a proton during rxn. Active form is deprotonated form. Increasing the pH will deprotonate more of the residue into the active form, increasing the rate of rxn.

Glucosidase rxn (acid-nucleophilic catalysis)

Acid-alpha-glucosidase is an enzyme that converts glucosylceramide to ceramide, a second messenger. Mutations in this enzyme cause Gaucher disease, where lysosomal storage is disrupted and causes defects in the liver, spleen, and neurological output. Enzyme has two Glu residues: - deprotonated one that acts as a nucleophile during glycosylation, remains nucleophile due to proximity of positive Arg residue - one that acts as an acid or a base, remains acid/base due to hydrophobicity driving up pKa of residue (8-10) * protonated catalytic acid to donate protons in the first step (glycosylation) * deprotonated catalytic base to abstract protons in the second step (deglycosylation).

Active sites complement TS

Active sites often complement the TS more than the substrate in order to stabilize this structure for rate acceleration, as well as to ensure the substrate does not permanently bind the enzyme. By complementing the TS, it encourages the rxn to proceed, rather than perfectly fitting the substrate and becoming inactive or not reacting.

Chymotrypsin optimal activity

Activity drops off above pH 8. Kcat dramatically increases above pH 7 due to deprotonation of His and Arg residues. When these residues are deprotonated, His can no longer perform its catalytic function. Arg is cleaved from the zymogen in the active environment, but if above pH 8, it will be neutral and therefore not targeted for cleavage. If the zymogen can not properly become the active enzyme, the substrate binding pocket is not formed properly. When pH is above 8, Ile residue is not properly protonated for interaction with binding pocket Asp residue, and the binding pocket cannot assemble properly.

Testing analogs

Add possible TS and substrate analogs to enzyme + substrate solution: - will be able to determine inhibitory ability - substrate will out-compete poor analogs Mutating the enzyme: - will still have original active site and microenvironment - but no diversity of active sites for testing, unlike with antibody production

Allosteric regulation

Allosteric activator/inhibitor binds at a site away from the active site, which alters the conformation of the enzyme to either allow or block substrate binding. Activators change the active site to fit the substrate. Inhibitors change the active site to block out the substrate.

Membrane binding domain

Allows enzyme to localize on the membrane where the substrate is found.

Velocity/concentration curve analysis

Analyzing these curves will reveal how fast an enzyme operates, how efficiently an enzyme converts substrate to product, and how an enzyme responds to stimuli or inhibitors. This may tell you if a reaction is likely to occur within a cell, and about any regulating factors.

Antibodies (cont'd)

Are catalytically dead: - have binding energy, desolvation effect, microenvironment, proximity effect, etc. - have no catalytic residues

Steady state approximation

Assumes that [ES] remains constant during velocity/rate measurements.

Studying single ionizing groups

Assumptions: - only one ionization state of enzyme is active - enzyme is stable over pH range being studied - rate determining step does not change with pH If group must be unprotonated: - velocity increases with increasing pH - group is a catalytic base If group must be protonated: - velocity decreases with increasing pH - group is a catalytic acid

Enzymes

Biological catalysts that are capable of performing multiple rxns (are recycled). They can be proteins or RNA, are faster than chemical catalysts, and act under mild conditions (bodily temperature/pressure). They are highly specific and tightly regulated.

Substrate reactivity

Bonds that will be broken and formed must have proper reactivity with the enzyme.

Metal ion catalysis (CONT'D)

Can be used to interact with AA sidechain groups in the active site to promote reactivity of those groups through electrostatic interaction. Catalytic activity is increased through proper active site orientation. EX. metalloenzymes, like urease, are important for maintaining proper protein structure and active site residues, maintain proper enzyme folding through interactions with AA residues Enzymes can also accept metal ions as co-factors that assist in activity. EX. kinases use Mg or Mn as co-factors in order to allow ATP to bind as a neutral molecule. Mn/Mg neutralize the negative charges on ATP.

Carbonic anhydrase rxn (metal ion covalent catalysis)

Carbonic anhydrase is an enzyme that catalyzes the conversion of CO2 and water to bicarbonate (reversible). A Zn molecule is coordinated with 3 His residues in the enzyme cleft. His residues drive coordination of Zn with ordered H2O 1. Zn coordinates with an ordered water molecule, releases H+ to leave -OH as a nucleophile. 2. CO2 binds to the enzyme active site, which positions it to react with -OH. 3. -OH attacks CO2, forming CO2- intermediate, stabilized by Zn. 4. Ordered water molecule binds to the active site, interacting with Zn to release bicarbonate as a product.

Kcat

Catalytic constant of the rxn. Also known as turnover number of the enzyme - max number of substrate molecules converted to product over time. Determined experimentally via Kcat = Vmax/[Et] where [Et] is the total enzyme concentration. Can only be determined this way for enzymes following MM kinetics.

Kcat/Km

Catalytic efficiency of the enzyme. Direct measure of the efficiency of the enzyme in transforming the substrate. Combines the effectiveness of transformation of the bound product with the effectiveness of productive substrate binding. Can be obtained from steady state experiments.

Electrostatic catalysis

Charged transition state is neutralized by an oppositely-charged group from the active site of the enzyme. Main amino acids that act as electrostatic catalysts: - Asp, Glu, His, Lys, Arg Electrostatic interactions in the active site (ion-pairs, salt bridges) can collectively attract substrate into the active site pocket to stabilize the transition state to reduce the activation energy.

EX. Chorismate mutase

Chorismate - predominant in small organisms, used to create amino acid residues. Generated various catalytic antibodies using different TS analogs as the antigen. Antibodies will mimic the active site microenvironment to support the TS or substrate. Steps in experiment: 1. Synthesize possible TS structure and substrate analogs 2. Animals are used to produce antibodies against each analog - each assigned a unique analog 3. Take antibodies and incubate with substrate in absence of enzyme 4. Document activity and product formation of each antibody ABs were able to exhibit catalytic activity. Those resembling the TS closest had the most activity and produced the most product.

Active site

Complements the structure of the substrate - most commonly the transition state of the substrate. Contains amino acid residues that function in substrate binding, chemical catalysis, and product release.

CX5R

Consensus motif of phosphatases. Cys is critical for activity. His usually comes before the Cys, and coordinates an ordered water molecule in the rxn. Arg is needed to stabilize the negative charges of the phosphate moiety. Ser/Thr usually occur after Arg. Help with stabilizing thiol-phosphate intermediate during rxn.

Motif

Conserved portion of a protein that does not have independent function or folding.

Domain

Conserved portion of a protein that is capable of independent function, folding, and stability.

Covalent catalysis

Covalent bonds are formed between an enzyme and its substrate during the formation of the TS. Bonding is usually initiated by an electron-rich group in that active site (nucleophile) that attack electron-deficient centers to form covalent intermediates. Involves a two-part reaction process that contains two energy barriers. This is because two TS are formed: one before the intermediate is formed, and one after.

Phosphatases

Dephosphorylate molecules. Can be Ser/Thr specific, Tyr specific, or dual specific. Goal is to induce protein conformational change by removing phosphate from AA sidechains. - can increase/decrease activity - reveal new binding interfaces - regulate and control binding - induce ubiquitination - control localization - change function

Competitive inhibition

Direct competition between a substrate molecule and inhibitor for the active site of an enzyme. - inhibitor binds same site as substrate - inhibitors only bind to free enzyme - structure of inhibitor and substrate/TS are often similar - most common inhibitor By forming EI complex, inhibitor reduces amount of free enzyme. After the ES has formed, inhibitor cannot affect ES.

Catalytic acid

Donates a proton during rxn. Active form is protonated form. Decreasing the pH will protonate more of the residue into the active form, increasing the rate of rxn.

Binding energy

Enzymes provide a docking site and microenvironment for proper substrate orientation for reaction. The binding energy from this interaction can be used for rate enhancement. Formation of the ES complex releases free energy, used to increase rate of rxn. Same effect as adding more catalyst. The ES complex has a unimolecular orientation that loses entropic penalties.

Transition state stabilization

Enzymes reduce activation energy by stabilizing the transition state structure. Often neutralize negative or positive charges in the TS. The catalytic residues of an enzyme are not crucial for this.

Microenvironment

Everything in and around the active site that allows catalysis of the substrate to occur. Contains a large number of hydrophobic residues to allow for interaction with the substrate.

Michaelis-Menten equation

Goal of equation is to calculate the rate of rxn at the steady state.

Chymotrypsin

Has a catalytic triad of Asp (used to increase His pKa, prevents rotation of His), His (activates Ser as a nucleophile through catalytic base abstraction), and Ser. Specificity is determined by the active site binding pocket, which is large and binds aromatic side chains, usually Lys. 1. Ser acts as a nucleophile, attacking the [ES] complex, producing an covalent acyl-enzyme intermediate. 2. His acts as a catalytic acid, donating a proton to the first leaving group (P1) 3. His acts as a catalytic base, abstracting a proton to activate an ordered water molecule. 4. Hydroxide acts as a nucleophile, attacking the acyl-enzyme intermediate to regenerate Ser. Chymotrypsin activity drops off above pH 8. Conformational changes necessary for substrate binding do not occur due to deprotonation of amine terminals, causing an increase in Km.

Antibodies

Hypervariable region - dictates the specificity or affinity for the molecule an antibody was produced against. When raised against an analog, it will mimic the active site of the enzyme the analog was for. Antibodies raised against the most correct analog should show the highest catalytic activity, but will not be as high as the enzyme since they are missing the catalytic residues.

Hammond's postulate ramifications

If species are sequential on the rxn coordinate and similar in energy, they are similar in structure. Unstable intermediates on the rxn pathway are predicted to resemble the TS structure. The structure of these can be obtained experimentally. Changes in structure that stabilize or destabilize intermediates will do the same to TS's that lead to them.

Ground state destabilization

Increase in energy, and decrease in stability, in going from one state to another. Sometimes occurs after enzyme-substrate binding.

Noncompetitive inhibition

Inhibitor binds to both free enzyme and ES, and not usually to the active site - binds at allosteric site. Only see product formation when the inhibitor is released. Induces a conformational change in the enzyme that affect catalysis. ESI complex cannot react. Results in reduced product formation, and cannot be overcome by increasing [S].

Measuring initial rates

Initial velocity is the amount of product produced per minute. Can be measured using an assay to observe absorbance with respect to time. Abs vs. time is then plotted and the slope of the tangent can be determined. Tangent is used to convert Abs to velocity using Beer's Law. Slope of the tangent is equal to the change in Abs over time, where the change is absorbance = e(delta[c])(L). If the change in [c] occurs over a known time period, then (delta[c])/time = initial v.

Rapid mixing techniques

Involves mixing two or more solutions together as rapidly as possible, and getting the solution to the optical observation point quickly. Faster mixing techniques means one can observe earlier into the reaction (small dead time - time from mixer to observational cell).

Competitive inhibition consequences

Km is increased - higher [S] is needed to reach Vmax. Increases by a factor of a - corrects for fraction of enzyme unavailable for catalysis due to inhibitor binding. Vmax is unchanged - at high [S] the inhibitor is displaced from the active site, ES formation is favored over EI.

Noncompetitive inhibition consequences

Km is unchanged since substrate can still bind the enzyme. Vmax is lowered by a factor of a - at high [S] the inhibitor is still bound to enzyme. Kcat is lowered by a. Additional substrate will not displace the inhibitor. Consequence is a lower turnover rate. Results in small conformational changes that affect the efficiency of catalysis. Affects the flexibility of catalytic residues by changing their orientation.

Vmax

Maximum velocity. Directly proportional to enzyme concentration.

Molar absorptivity (e)

Measure of the ability of an analyte to absorb light at a specified wavelength. Beer's Law is only valid when e is a constant.

Km (Michaelis constant)

Michaelis constant. The concentration of substrate [S] needed to reach half max velocity. Measure of substrate affinity. Km = [S] when V = 1/2Vmax A lower Km value indicates a higher affinity for the substrate. At high [S], initial velocity = Vmax. At low [S], can only estimate initial velocity. Km can change with temperature, pH, pressure, and inhibitors.

Orotidine 5'-monophosphate decarboxylase

Model enzyme: - mediated by microenvironment - positioning of CO2- group close to negatively charged Asp group so repulsion will aid in bond breakage - ground state destabilization. Phosphate is critical for proper positioning: - binds to phosphate binding pocket through interaction with ordered H2O and Arg * H2O provides stabilizing force with strong H-bond interactions * Arg residue is conserved, uses positive charge to drive strong electrostatic interaction with P - binding forces positioning of rest of substrate, forces CO2- close to charged Asp Carbanion stabilization: - may be through electrostatic interaction with a positive charged residue - Lys may act as a catalytic acid to stabilize the negative C by shielding negative Asp charges or donating a proton

Inhibitor modelling

Modelled after the transition state: - more specific so a lower dosage can be used - less off-target effects due to specificity Allosteric sites are not as good to use since they are not as specific and can have more off-target effects.

Substrate affinity

Must bind relative well, but not have a perfect fit. Binding is through H-bonds, electrostatics, and hydrophobic interactions. If substrate fits perfectly, the enzyme will not convert and release the substrate, which inactivates the enzyme.

Applications for active site inhibitors

Obtaining mechanistic info for an enzyme-mediated rxn: - determine cellular phenotype following enzyme inhibition Drug therapy: - inhibit overactive mutant enzyme - inhibit normal enzyme that functions in an opposing pathway to a mutated loss of function enzyme/pathway

Acid-base catalysis

Occurs very slowly without an enzyme to stabilize transition state. Concerns the donation or abstraction of protons from catalytic acids or bases. Main amino acids that can act as acid-base catalysts: - Asp, Glu, His, Lys, Cys Acid-base rxns tend to be controlled by the pKa of an amino acid sidechain, which can be altered through pH to alter catalysis. pH-rate profiles can be used to distinguish between acid and base catalysis, lead to identification of catalytic residues involved.

Studying two ionizing groups

Often there are two groups that need to be in correct ionizable form for proper activity. Ex. protonated Asp as a catalytic acid and unprotonated Cys as catalytic nucleophile Velocity vs. pH plot will result in a bell curve shape. Highest point is the pH optimum of the enzyme for best activity of each residue. pH dependency is not dependent on the concentration of [S], but on the residues. Can do mutagenesis experiments to change pH dependency and determine residues critical for pH dependency.

Detecting chymotrypsin intermediates

Para-nitrophenylacetate attached to phenylalanine used as a synthetic substrate for chymotrypsin. Para-nitrophenol is chromophoric and can be tracked through a rxn.

Catalytic domain

Protein domain that contains the active site and allows for proper folding of entire structure. Stabilizes the active site to form a key microenvironment. Serves as site of regulation, can hold an allosteric site, maintains the distinguishing features of enzymes.

Proximity effects

Rate increase due to two reactants being brought out of a dilute environment and being placed close together. Made possible by the catalytic domain. Include: - increase in effective concentration (same effect as formation of ES complex) - binding energy - desolvation

Transition state theory

Reactants pass through high-energy transition states before forming products. Reactants and products are usually stable in their ground states.

Desolvation

Removal of the substrate from water or other solvents, or dehydration of substrate. Eliminates the energy barrier imposed by ordered solvent molecules that would slow a rxn. This results in an accelerated rxn.

Activation energy

Represents the different in energy between the reactants and transition state(s). The higher the activation energy, the slower the rxn will proceed. Enzymes accelerate rxn rates by reducing activation energy. They do not affect the Gibbs energy of the reaction, and have no effect on the equilibrium.

Increase in effective catalytic group concentration

Result is an increase in the effective concentration of catalytic groups in an enzyme. Effects: - increases chances of a rxn since molecules are close together in an ES complex - ES complex is an intramolecular entity that reduces entropy loss

Types of active site inhibitors

Reversible inhibitors - bind non-covalently so that they can be displaced and do not permanently deactivate an enzyme. Irreversible inhibitors - bind covalently to the enzyme and cannot be displaced, so they permanently deactivate the enzyme.

Why study pre-steady state?

SS cannot give information on individual steps, or on the rate of steps that are faster than the rate-determining step.

Cooperativity regulation

Seen in enzymes with multiple active sites. Active form of an enzyme is stabilized by the binding of one substrate molecule, increasing its affinity for more substrate molecules to bind. First binding changes the conformation in such a way that the enzyme can accommodate more substrate.

Velocity/concentration curve

Shows the progression of a rxn as a function of velocity. Without an enzyme, the rxn rate is proportional to [S]. With an enzyme, a curve is observed. 1. It presents with first order rate kinetics where the substrate binding is rate limiting. If substrate does not bind, the rxn does not proceed. 2. Rate rises with increasing [S], but is not directly proportional. 3. Presents with zero order rate kinetics where an increase in [S] has little effect on the rate due to enzyme saturation. Rate limiting step is the conversion of substrate to product to free up more enzyme active sites.

Metal ion catalysis

Specific type of electrostatic catalysis. Uses positively charged metal ion to stabilize negative charges in the TS or intermediate to increase the rate of catalysis. Stable charges aren't affected by temperature, pH, etc. They act as electrophiles to stabilize nucleophilic attack. EX. Coordinating a Cobalt complex increases the ability of a nucleophile to catalyze hydrolysis of glycine ester through stabilization of negative TS charges.

Enzyme kinetics

Speed of and measurement of a rxn. Important for determining the efficiency of the rxn. Characterizing the rate or velocity of an enzyme catalyzed rxn. Key is understanding that rxn velocity is altered by changes in substrate concentration. E + S --> ES --> E + P E + S --> ES --> EI* --> E + P2 --------------| -------------P1

pH effects on enzyme activity

Structural: - extreme changes will denature folded structures due to repulsive forces - milder changes can dissociate oligomeric state of enzyme into inactive monomers by disrupting hydrophobic oligomeric dimerization - can use fluorescence to determine effects on protein's native folding Catalytic: - substrate ionization - affects enzyme-substrate productive binding - ionization state critical for catalysis since catalytic domain contains acid/base groups Km concerns productive substrate binding through ion pair, H-binding, and hydrophobic interactions. Kcat concerns chemical transformation through acid/base, nucleophilic, and electrostatic catalysis.

Analogs

Structurally resembles transition state and stabilized due to bonds. - unable to be acted upon since they have bonds that cannot be hydrolyzed - good for irreversible inhibition - higher activity seen with TS analogs than substrate analogs

Inhibitors

Substances that fit and bind an enzyme, but do not have the proper reactivity.

Stopped-flow method

Syringes are filled with enzyme or substrate solution and compressed to express small amounts of each into a mixer, then stopped. Once flow of E and S is stopped, the solution is allowed to age in the mixer, which can be controlled. By forcing the flow into the optical detection cell, you can see the solution at different ages. Has low dead times, usually around 1 millisecond or less. Is very expensive, and cannot accurately identify species. Can monitor a number of enzyme-substrate complexes during the rxn by looking at large wavelength and time ranges. Single wavelengths can be selected and Abs vs. time measurements conducted to obtain the rate of formation and decomposition of specific rxn intermediates. Measure Abs vs wavelength at different time ranges and dead times. Can be used to see different intermediates and how fast they form. - can look for interference - determine max wavelength - dynamic in real time due to wavelength scan variance based on time range used - seen as peaks on a graph

Enzyme specificity

The active site dictates specificity: - specific for one type of chemical group - substrates interact in stereospecific manner (must fit) - substrates bind relatively well -substrates must react

Lineweaver-Burke plots

Transform the Michaelis-Menten equation from a curved line to a straight line for better accuracy when studying kinetics. Useful for studying enzyme inhibition.

Hammond's postulate

Two states occurring consecutively after one another during a reaction, having nearly the same energy content, will be similar in structure. The TS will resemble the reactants, intermediates, or the products more depending on which state is closer to it in energy.

Ex. Gleevec

Tyrosine kinase inhibitor used to treat chronic myelogenous leukemia. CML involves uncontrolled proliferation of myeloids (white blood cells). Prominent feature is a chromosomal translocation event involving chromosomes 9 and 22, forming a BCR-ABL Philadelphia chromosome. Expresses a chimeric tyrosine kinase. ABL becomes constitutively active, has a critical residue that controls how the cell turns on or off. Gleevec was developed from a lead compound designed to inhibit all tyrosine kinases. Inhibits BCR-ABL by binding to its ATP binding site. It prevents phosphorylation of substrates involved in proliferation, resulting in growth arrest and apoptosis of affected cells

Altering pKas

Use Glu or Asp: - can lower pH in the microenvironment or alter pKa of specific amino acid residues - create a repulsive situation that forces a residue to keep its proton and drive up the pKa (usually when placed together)

Use of mechanistic/biological studies

Use of RNAi to knockdown and/or gene knockout: - see overcompensation by the cell - uses production of similar gene family members to restore function of non-functional gene Cell has difficulty compensating for inhibitors: - targets already translated protein

Structural biology of enzymes

Use of x-ray crystallography to solve the structure of enzymes. Can use with or without substrate provides functional and biological info. Can be used to identify amino acids involved in chemical catalysis.

Measuring acylation rate of chymotrypsin

Used a modified stop-flow with chromophoric inhibitor displacement with proflavin. Proflavin is a competitive inhibitor of chymotrypsin (has aromatic groups that bind active site), and absorbs strongly at 465nm when bound. Cannot bind to acyl-enzyme intermediate. Syringe 1 = acetyl-Phe ethyl ester Syringe 2 - chymotrypsin-proflavin solution Syringes are compressed to mixer and allowed an equilibration time. This showed an initial rapid displacement of some proflavin in the dead time. As acyl-enzyme int is formed, the equilibrium breaks down. Proflavin is completely displaced, resulting in a decrease in 465nm absorbance. ** rate of acyl-enzyme formation (acylation) = rate of Proflavin Abs decrease Absorbance is constant until acyl-enzyme breakdown. Proflavin can rebind to enzyme, and 465nm Abs increases. ** rate of acyl-enzyme breakdown (deacylation) = rate of Proflavin Abs increase Acylation rate is obtained from exponential second phase of Abs decrease.

Spectrophotometry

Used to determine the concentration of a product over a period of time. Done by using Beer's Law, which describes a linear relationship between the absorbance and the concentration.

Spectrophotometer

Used to measure the amount of light a sample absorbs. Operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.

Enzyme inhibition

Useful for learning mechanistic details of enzyme-mediated rxns and physiological properties of enzymes. Useful for understanding how enzymes may be regulated using natural cellular inhibitors. Useful for drug therapy - most are analogs used as inhibitors.

Quenched-flow method

Uses a chemical or physical quencher to freeze a rxn at a certain time point, usually with acid (denatures the enzyme) or a deep freeze. Each moment can be analyzed by analytical techniques to determine the identity of enzyme intermediates. Aging hose length can be varied to see different ages of the rxn. Enzyme can then be denatured to release the intermediate for study, using urea, etc. Can also radiolabel the substrate to monitor and trap at different time points. Good option for rxns that do not have chromophoric groups (fluorescent) or can't be monitored by Abs levels. Longer dead times of 100 ms or longer. Usually combined with stopped-flow techniques. Use NMR or circular dichroism to follow the enzyme's conformational changes.

Enzyme regulation

Usually tightly regulated through several means. Unregulated enzymes can become constitutively active or inactive, and can disrupt cell homeostasis, leading to disease states.

Regulation of downstream signalling by RTKs

When a ligand binds RTK surface receptors, RTK dimerizes and is able to phosphorylate molecules that trigger downstream signalling. When PTP is reduced, it is able to block RTK phosphorylation and therefore downstream signalling. When PTP is oxidized, it is unable to do so and signalling can occur. PTP is oxidized through the production of hydrogen peroxide produced by NADPH oxidase, which is activated by RTK.

Catalytic competence

When the active site is in the correct orientation for catalysis to occur.