Lectures 22-23 biochem sad day sad day

kcat

turnover number, the maximum number of substrate molecules converted to product per enzyme active site per second

kcat/Km

Catalytic efficiency; describes how effectively an enzyme processes substrate, particularly at low substrate concentrations

Oxidoreductases

Electron transfer

Transferases

Group transfer

Hydrolases

addition of H2O

Lyases

Bond cleavage without hydrolysis or electron transfer

Racemases and Epimerase

Isomerizations

Ligases

Bond formation

Catalysis

What can destabilizing the substrate ground state can promote?

Electrostatic stabilization

Favorable interactions between charges or dipoles that lower the free energy of a particular state

Induced dipole

A redistribution of electron density caused by a nearby charge or electric field

transition state

An enzyme active site is designed to favor the shape and charge distribution of the

The active site provides favorable interactions with the transition state’s shape and electrostatic distribution, reducing the activation barrier

Why does an enzyme stabilize the transition state more effectively than the substrate?

Transition state analog

A stable molecule that resembles the transition state of an enzyme catalyzed reaction

transition state

The enzyme’s active site is optimized for the

molecules that resemble the transition state can bind more tightly than ordinary substrates

what does optimization for the transition state get you?

inhibition constant, used to describe inhibitor binding

What does KI mean?

Stronger inhibitor binding

the smaller the KI

substrate affinity measure

The transition state analog binds extraordinarily strongly relative to the

the transition state analog is a very potent, tight-binding enzyme inhibitor

what does a large Km/Ki ratio mean?

Near attack complex

Configuration in which reacting groups are positioned at favorable distances and orientations for chemical reaction

It increases the probability of productive reaction by organizing the reactants into favorable positions and orientations

What is the advantage of a near attack complex?

Proximity effect

Increased reaction rate because reacting groups are held near one another

Orientation effect

Increased reaction rate because reacting groups are aligned appropriately

effective concentration

The locally enhanced availability of one reacting group to another when they are positioned together

Orbital

correct —- alignment is essential for efficient bond formation and bond cleavage

Covalent catalysis

occurs when an enzyme temporarily forms a covalent bond with a substrate during the reaction

Covalent catalysis

in ——- the enzyme directly participates in bond formation rather than only positioning the substrate

Electrophile

Electron deficient species that accepts an electron pair

Nucleophile

Electron rich species that donates an electron pair to form a bond

Electrophiles

Protons, metal ion, carbonyl carbons, and cationic imines are examples of

Nucleophiles

Hydroxyls, sulfhydryls, amino, imidazole are all examples of

covalent catalysis

this is an example of

Low-energy barrier hydrogen bond

A hydrogen bond in which the barrier to proton movement between donor and acceptor atoms is unusually small

Proton transfer

The distance between proton donor and acceptor atoms can influence the barrier for

Proton transfer is relatively difficult

Normal hydrogen bond- high energy barrier

Proton transfer becomes easier and potentially faster

Low barrier hydrogen bond- reduced energy barrier

There is no central barrier separating two proton positions

Single-well hydrogen bond- essentially no central barrier

Specific acid-base catalysis

Catalysis involving H+ or OH-

General acid-base catalysis

Catalysis involving other proton donors or acceptors, such as buffer molecules

Specific acid base

what acid base graph is this showing?

General acid base

what acid base graph is this showing?

General base

A species that accepts a proton during a catalytic step

General acid

A species that donates a proton during a catalytic step

Imidazole facilitates hydrolysis of p-nitrophenyl acetate by accepting a proton

Uncatalyzed keto-enol tautomerization

Acid catalyzed keto-enol tautomerization

(electron withdrawing)

Base catalyzed keto-enol tautomerization

(electron donating)

most enzyme is protonated, 50% is protonated, most enzyme is deprotonated

at low pH what happens? at pH=pKa? at high pH?

less abundant

The reaction rate decreases as the protonalted active form becomes

protonated, deprotonated

Some enzymes require one group to be —— and another to be ——

a graph of enzyme activity and pH

what is this graph showing?

The catalytic groups must have specific protonation states, and those states are most favorable within a particular pH range

Explain why enzyme activity may be low at both acidic and basic pH

Lysozyme

an enzyme that hydrozyles polysaccharide chains in certain bacterial cell walls

Lysozyme and pH dependence, supporting involvement in ionizable catalystic groups

What does this show?

Mass spectrometry

what does this image show?

Mass spectrometry

analytical technique that measures mass-to-charge ratios (m/Z) of ions

D52

What forms a covalent adduct with the substrate

The mechanism involving a covalent enzyme-substrate intermediate formed by Asp52

Which mechanism is supported for HEWL?

Chymotryspin

A serine protease that catalyzes hydrolysis of peptide bonds

Chymotryspin: covalent and acid base catalysis

What is this showing?

enzyme substrate intermediate

Fast reaction kinetics provide evidence for a temporary covalent

fast formation of an acyl enzyme intermediate

Initial rapid burst corresponds to

intermediate adn enzyme regeneration

The slower phase reflects subsequent processing of the

Neutron diffraction

How can you find which group accepts the proton from serine during catalysis?

Neutron diffraction

Determins structural information using neutron scattering by atomic nuclei

Mechanism involving covalent catalysis with acid base assistance

What does this show?

equilibrium for free molecules

The equilibrium between enzyme bound substrates and enzyme bound products can differ from the

substrate and product species have equal intensities, supporting equilibrium constant near 1

what does this NMR signal show

Metals

What provides chemical capabilities that amino acid side chains alone may not supply?

Heme

Iron containing prostehtic group that participates in electron transfer and oxygen binding

HEME

what structure is this showing?

FADH+

what does this show?

FADH2

What does this show?

They can participate in both ratical and hydride equivalent chemistry

Why are flavins versatile in redox reactions?

NADPH

what is this showing?

NADH

What is this showing?

Pyruvate Dehydrogenase Complex

what is this showing?

Cobalamin

a colbalt containing cofactor involved in certain rearrangement reactions

Cobalamin

what diagram is this illustrating

Pyridoxal phosphate

what is this showing?

Pyridoxal phosphate

a vitamin B6 derived cofactor that facilitates amino acid chemistry by stabilizing reactive intermediates

It stabilizes otherwise unfavorable carbanion like intermediates through resonance

Why is PLP useful in amino acid reactions?

HIV protease

— contains flexible flaps that msut move to alllow substrate entry and product release

NMR spectroscopy

what can help identify protonation states of catalytic groups?

NMR chemcial shifts, investigating catalytic asparte residues

what graph is this showing?

Liver alcohol

a dehydrogenase uses zinc to faciliate hydride transfer

Zinc

Stabilizes the developing negative charge on oxygen and facilitates hydride transfer to the carbonyl carbon

Carbonic anhydrase

catalyzes reversible interconversion between carbon dioxide and bicarbonate

Electron tunneling

quantum mechanical process that allows an electron to pass through an energy barrier taht would be forbidden by classical mechanics

Electron transfer rates

— depend on the distance between electron donors and acceptors

quantum tunneling

electron transfer can occur by