Exam 3 (lec 16-22)

Bisubstrate rxns

• 2+ substrates

• Single displacement - random or ordered

• Double displacement

• >60% of biochem rxns

Single displacement Random

• both substrates bund ro enzyme b4 product is formed

• AEB to PEQ is rate limiting

creatine Kinase- random SD

• direction determined by [] of ATP, ADP, Cr and CrP and eq constant

• Most kinases have this mechanism

Single Displacement Ordered

• AEB to PEQis rate limiting

• ordered release of products

• no B will bind to E in the absence of A

• no turn over until both substrates bind

Single Displacement Bisubstrate

• saturation when curve no longer shifts

• resembles noncompetitive inhibitors

• higher V max

• increasing concentration = lower slope

Double Displacement

• 2 substrates bind and react separately in a ping pong manner

• burst kinetics w P being seen before Q

• E’ covalently modified enzyme intermediate

• product of enzyme’s rxn w A is released prior to rxn of enzyme w B

• sub bind independently to 1 another

Double Displacement Bisubstrate

• resembles uncompetitive inhibitors

• slope is constant

• increasing saturation = lower slope

Glutamate:aspartate Aminotransferase

• enzyme conforming to a double displacement bisusbtrate mechanism

Kinetic analysis

• Lineweaver-Burk plot to distinguish if mechanism is

  single or double displacement

• parallel = double

• non-parallel = single

Substrate binding assay

• to determine if substrate addition is random or ordered

• macrocalorimetry

• no heat = ordered, specificity

• heat = random

Exchange reactions

• attempt reverse reaction in absence of one substrate

• double displacement will allow the reaction

• single displacement will not

Exchange reactions example

• Sucrose phosphorylase and Maltose phosphorylase both cleave disaccharides and add Pi to one product

• Mix 32P tracers with G-1-P and add either enzyme…

Sucrose phosphorylase does catalyze the exchange

• DOUBLE DISPLACEMENT

Maltose phosphorylase does not catalyze the exchange

• SINGLE DISPLACEMENT

transition state (X‡)

• all rxns go thru

• short-lived, unstable

• high free energy and at least 1 partial bond

• NOT AN INTERMEDIATE

• rxn rate : [reactant that X‡

Enzymes lower ΔG‡

• enzymes bind to transition state more tightly than substrate

• lower free energy

• Don’t want Ks to be too tight as the enzyme needs to bind to X better than S (Kt)

X‡ needs to be stabilized more than S

• loss of entropy = ES needs to be destabilized

How ES is destabilized

• structural strain

• desolvation

• electrostatic effects

structural strain

• occurs bc of induced fir binding

lock and key hypothesis

• 1st explanation for specificity

induced fit hypothesis

• more accurate description of specificity

• favors formation of transition state

Destabilization of ES

• charged groups are desolvated and more active

• unfavorable interactions between like charges

How do enzymes work

• by speeding attainment of Keq, from 10⁷ to 10¹⁴ times faster

• Keq is not changed

• delta Grx is not changed

• But activation energy is lowered

Binding of Transition-State Analogs

• Kt may be 10^-20 to 10^-26 M (very tight)

• TSAs are stable mlcs that are chemically and structurally similar to transition sates

• act as competitive inhibitors and make good drugs

• outcompete substrates

Would TSAs bind better or worse than competitive inhibitors?

• better

• enzymes have a higher affinity to transition state than substrate

TSA in medicine

• Enalapril and Aliskiren lower blood pressure

• statins lower serum cholesterol

• protease inhibitors are AIDS drugs

• Juvenile hormone esterase in insects is a pesticide target

• tamiflu is a viral neuraminidase inhibitor

Statins

• like lipitor

• powerful cholesterol lowering drugs

• they are TSAs of HMG-CoA reductase, key enzyme in the biosynthetic pathway for cholesterol

• prolonged use = neurological deficits

Tamiflu

• neuraminidase inhibitor and antiviral agent

• based on the transition state of the neuraminidase reaction

Neuraminidase

• major glycoprotein on influenza virus

• essential for viral replication and infectivity

curved arrow

• shows movement of an electron pair from a filled orbital to an empty one

full arrowhead

• represents an electron pair

half arrowhead

• represents a single electron

bond-breaking event

• the arrow begins in the middle of the bond

Nucleophiles

• nucleus loving

• electron rich groups that can donate a pair of electrons

Electrophiles

• electron poor groups that can receive electrons

leaving group

• anything attached to the electrophile b4 rxn

Proton Transfer

• can change a nucleophile into an electrophile

protonation state of substrate and active site residues

• dependent on pKa and pH

• Histidine

pKa values

• can change in active site microenvironment

Mechanisms of Catalysis

1. catalysis by proximity (or formation of near-attack

   complexes)

2. Covalent catalysis

3. General acid-base catalysis

4. Metal ion catalysis

5. Low barrier hydrogen bonds

Catalysis by Proximity

• enzymes bring substrates in active site close enough and in the proper orientation so collisions occur at higher frequency

active site is preorganized into these

• near attack complexes (NACs)

• reacting atoms are in van der waals contact at an angle resembling the bond in the transition state

Near-attack Complexes

• characterized as having reacting atoms w/in 3.2 A and an approach angle of +- 15o of the bonding angle in transition state

Proteins are constantly moving

• bonds vibrate,

• side chains bend and rotate,

• backbone loops wiggle and sway,

• whole domains move as a unit

Active site conformation changes can:

• Assist substrate binding

• Bring catalytic groups into position

• Induce formation of NACs

• Assist in bond making and bond breaking

• Facilitate conversion of substrate to product

Covalent Catalysis

• active site residues form a temporary covalent bond w substrate

• bond is broken to regenerate enzyme

• occurs due to nucleophilic attack by AA side chains on electrophilic groups on substrates

• can involve prosthetic groups

• facilitates electron transfer

• most have unknown mechanisms

E’

• covalently modified enzyme intermediate

• double displacement

Trypsin , Chymotrypsin

react w serine

Glyceraldehyde-3-P dehydrogenase

react w cysteine

Phosphoglucomutase

react w serine

phosphoglycerate mutase, Succinyl-CoA synthetase

react w Histidine

Aldolase, Pyridoxal phosphate enzymes

react w Lysine and other amino groups

General Acid-Base Catalysis

• transfer of a proton in transition state

• facilitates proton transfer (back and forth)

• specific = H+ or OH- that has diffused into the active site

Transferring an H+ in acid-base catalysis may:

• activate nucleophiles

• stabilize charged groups

• improve electrostatic interactions that stabilize TS

Candidate amino acids for acid-base catalysis (Catalytic triad)

• Glu

• Asp

• His

Histidine Often Plays a Role

• might normally be protonated

• can be deprotonated by another group and act as a base, accepting protons from substrate

Water Often Plays a Role

• H2O can act as acid/base at active site thru proton transfer w an assisting active site residue

Shifts in pKa for Residues in Active Sites Create Catalytic Agents

• microenvironments play role in pKa of active sites

• primary: His, Cys, Asp, Glu, Arg, Lys, Tyr, Ser, Thr, Asn, Gln

Secondary Roles

• ~1/2 of AA engage directly in catalytic effects in enzyme active sites

Other residues may function in secondary roles in the active site:

• Raising or lowering catalytic residue pKa values

• Orientation of catalytic residues

• Charge stabilization

• Proton transfers via hydrogen tunneling

 Metal Ion Catalysis

• eg: zinc, magnesium, iron

• metal atoms lose electrons easily and exist as cations

• basics

Positive charge allows metal atoms to:

• stabilize transient and intermediate structures

• assist in forming strong nucleophilic species (image)

• hold substrate inside the active site

• stabilize charge

Low-Barrier Hydrogen Bonds (LBHBs)

• As distance between heteroatoms becomes smaller <.25nm, H bonds become stronger (almost covalent in nature)

• stabilization energy can approach 60kJ/mol in solution

• pKa values of the 2 electronegative atoms must be similar (Asp and His)

energy released in forming an LBHB

• can assist catalysis

typical H-bond strength

• 10-30 kJ/mol

typical O-O separation

• .28 nm

LBHBs

• .25 nm to <.25 nm

Trypsin, chymotrypsin, elastase

• igestive enzymes secreted as proenzymes/zymogens

Thrombin

blood clotting cascade

Subtilisin

bacterial enzyme

Plasmin

cleaves fibrin polymers of blood clots

Tissue plasminogen activator

cleaves plasmin proenzyme plasminogen and is administered to prevent heart attack

Acetylcholinesterase

NOT a protease but mechanistically similar in breaking down acetylcholine

Similar sequences, structures and mechanisms

evolutionary relatedness

Trypsin, Chymotrypsin and Elastase function

• proenzymes/ zymogens

• all cleave polypeptide chains

• identical sequences/aa are conserved for functionality

Structure of chymotrypsin

• His is flanked by Asp and Ser

• Ser is close to the peptide that would be cleaved in the rxn

The Catalytic Triad of the Serine Proteases

• catalytic triad at the active site of chymotrypsin

• His, Ser, Asp

Trypsin, Chymotrypsin and Elastase specificities

• similar structures and mechanism, but DIFF specificities

• Trypsin- C side of basic (EXCEPT WHEN FOLLOWED BY PROLINE bc of fixed phi angle)

• Chymotrypsin- C side of aromatic

• Elastase- C side of small, neutral residues

DIFF specificities

• given by active site

Enzymatic Fragmentation Trypsin

• 4 cut sites = 5 peptides

• product is a mix of peptide fragments w C terminal Arg/Lys + single peptide from the C terminal end of the polypeptide

Trypsin  Substrate-binding Pocket

• cuts after basics

• negatively charged

Chymotrypsin Substrate-binding Pocket

• cuts after aromatics

• hydrophobic (ser)

• rings themselves are hydrophobic

Elastase Substrate-binding Pocket

• cuts after small mlcs

• bulky residues like Thr and Val at top and shallow

Chymotrypsin Kinetics

• speed

• assayed using artificial substrate

• useful as nitrophenolate product absorbs at 400nm

• observed burst kinetics, suggesting first fast step second slow step

Serine Proteases Display Burst Kinetics

• quick cut

• slow serine binding

Serine Protease Mechanism

• mix of covalent + general acid-base catalysis

• Asp orients His and forms LBHB

• His acts as acid/base

• Ser forms covalent bond w peptide to be cleaved

• trigonal C into tetrahedral C

• Tetrahedral oxyanion intermediate stabilized by backbone NH groups of Gly + Ser

Transition-State Stabilization

• chymotrypsin involves 2 tetrahedral oxyanion transition states

• these are stabilized by a pair of amide groups termed oxyanion hole

the oxyanion hole

• O- in tetrahedral oxyanion is stabilized by interaction w the backbone NH groups of Ser + Gly

Catalytic Triads In General

• found in several hydrolases + transferase enzymes

• occur via divergent + convergent evolution

• Residues far away in primary structure

Catalytic Triads Include:

1. cid that orients and stabilizes the base (Asp, Glu, His)

2. Base that polarizes the nucleophile ( His, rarely Lys)

3. Nucleophile that attacks substrate (Ser, Cys, sometimes Thr)

Aspartic Proteases

• DIFFERENT structure and mechanism than serine proteases

• active site = 2 Asp

• cleave peptide bond btw 2 hydrophobic aa

• No covalent catalysis

Structures of HIV-1 protease and pepsin

• HIV-1 protease - homodimer

• pepsin- monomer

• Each lobe contributes one catalytic aspartate to the active site.

pH-dependence of asp proteases

• mechanism requires that one Asp be protonated and the other Asp be deprotonated when the substrate binds

• active at acidic pH

A Mechanism for the Aspartic Proteases

• LBHB allowing “hydrogen tunneling)

• LBHBs play a role in states E, ES, ET’, EQ’, and EP’Q.

Protease Inhibitors for AIDS Patients

• HIV-1 protease selectively inhibited = new HIV particles cannot form

• structure based drug design has developed several inhibitors

HIV-1 Protease

• novel asp protease

• cleaves polyprotein products of HIV genome

• remarkable imitation of mammalian asp proteases

• homodimer- more genetically economical

• active site = 2 fold symmetric flaps

Protease Inhibitors Block the Active Site of HIV-1 Protease

HIV-1 protease complexed with the inhibitor Crixivan (red)

made by Merck. The “flaps” that cover the active site are

green; the catalytic active-site Asp residues are violet.

enzyme regulation overview

• rxns to match cell requirements

• natural physiological phenomenon

• focus on activity

Achieved by regulating enzyme:

1. abundance

2. activity

abundance

• controlled by gene expression (not treated)