Section 5 + 6- Oxygen Transfer Proteins and Zinc Metalloenzymes Flashcards
How is energy produced?
Respiration in which O2 is reduced to water
What protiens help energy production?
- enzymes
- reactions up to 10^12 times faster than model
How is oxygen normally activated?
- by coordination to a metal centre
- iron or copper are common
- increases rate of reaction
What are Cytochromes P450?
Where are they found?
Why 450?
- a family of metalloenzymes
- function as monoxygenases - found in mammalian tissues, birds, fish, plants, insects, yeasts, bacteria,
- name from CO adducts characteristic absorption bands at 450 nm.
What are oxygenases?
- enzymes that inset oxygen into other molecules
- a monooxygenase inserts one oxygen
- deoxygenate inserts two oxygens
What does Cytochromes P450 catalyse?
- aromatic and aliphatic hydroxylation reactions
- other oxidation reactions
- essential for biosynthesis, metabolism and detoxification
How does a reaction with Cytochromes P450 work?
- One oxygen atom is inserted
- One oxygen atom is reduced to H2O by a reducing agent
What is the general structure of Cytochromes P450
- Similar to Hb and Mb
- Based on a Haem Ring
- Iron coordinated by S(Cys)
Give the steps of the Cytochromes P450 catalytic cycle
- cytochrome P-450 contains a low-spin Fe(III) centre with an addition ligand, probably water, in the sixth coordination site
- RH binding and H2O loss to give high-spin Fe(III) state (2). Redox potention changes from -300 to -170 mV
- One electron reduction to high-spin Fe(II) (3)
- Binding of O2 and one-electron transfer from Fe(II). ν(O-O) = 1140 cm-1
- Acceptance of electron to give peroxy state (5) (rate limiting step)
- Protonation to give hydroperoxy state (6)
- Further protonation leading to heterolytic O-O bond cleavage and generation of a
formally oxoiron(V) species or Fe(IV)=O species with the porphyrin ring existing
as a radical cation (7)
- Oxygen atom transfer from (7) to substrate to give alcohol product and
regeneration of resting state
What happens during a peroxide shunt?
Cytochromes P450
ingle oxygen atom donors such as hydrogen peroxide, alkyl hydroperoxides, peracids, and iodosylbenzenes can also be used to generate oxygenated products by bypassing the intermediates (3)–(5).
Why is methane hard to hydroxylate?
- High C-H bond energy
- No dipole moment
- No additional functionality to assist in binding in protein active site
How is methane converted to energy?
Where is this useful?
- using MMO (methane monooxygenase)
- important in methanotrophic microorganisms where methane is the sole energy source
What is the reaction for MMO?
CH4 + O2 + NADH + H+ + MMO goes to CH3OH+H2O+NAD
What is NAD?
The coenzyme for MMO
What is the structure of the hydroxylase component of MMO?
- 2 Diiron centres
- Bridging hydroxo group
- Fe(III) resting state for both irons
Describe the MMO catalytic cycle
- Reduction to diferrous Fe(II)state
- O2 addition to give peroxy intermediate
- Proton donation to outer oxygen of peroxy-ligand, leading to H2O elimination
and generation of a Fe(V)-Fe(III) state [or Fe(IV)-Fe(IV)] - Fe(V) accepts a hydrogen from methane to give a CH3 radical and Fe(IV)-OH
5.Fast generation of methanol via donation of OH
What is Tyrosinase?
- Tyrosinase is a monooxygenase enzyme which catalyses the hydroxylation of monophenols to diphenols
- can also act as a two-electron oxidase catalysing the oxidation of diphenols to quinines.
What is the structure of Tyrosinase?
- Dinuclear site with two Cu(I) centres
- Like Hc
- O-O streching frequency of 755 cm-1
Describe the catalytic cycle Tyrosinase
- Addition of oxygen to give di-Cu(II) oxy form
- Coordination of phenolic substrate in axial position
- Ortho-hydroxylation of the phenol
- Intramolecular electron transfer to give quinone product and regenerate
active site
What are the uses of Zinc?
- catalytic processes
- structural engineering of proteins
- regulation processes
How does zinc exist under physiological conditions?
- dicationic Zn(II) - closed shell d10 configuration
- diamagnetic and colourless
What are the important characteristics of Zn (II) in biological systems?
- Strong Lewis acid.
- NOT redox active.
- Ready formation of low coordinate binding sites (> acidity)
- Accessible coordination numbers of 4, 5, 6.
- Easily deformed coordination geometry.
- Easily undergoes mono-ligand substitution chemistry
What is a unique feature of monzinc sites?
- water molecule that can be activated by ionisation, polarisation or be ready for dislacement
How does Zn coordination change the pKa of water?
free H2O - pKa = 15.7
metal bound in [Zn(H2O)6]2+ - pKa ≈ 10
metal bound in [ZnL3(H2O)] - pKa ≈ 7
the lower the coordination number of the zinc the more acidity there is for the attached water
How is the ‘transparency’ of Zn(II) overcome?
- replacing Zn(II) with Co(II) which has a similar ionic radius and can tolerate similar coordination environments.
- often possible to replace Zn2+ in a protein by Co2+ without greatly perturbing the protein conformation.
- As Co2+ is a d7 metal centre, its UV spectra can be used to monitor its coordinate geometry and changes in the UV spectra with pH can be used to study the deprotonation of any attached H2O molecules.
Where is carbonic anhydrase (II) found?
What does it catalyse?
- found predominately in red blood cells
- catalyses the reversible hydration of CO2:
CO2 + H2O goes to HCO3- + H+
Why is CO2 hydration important?
- removal of CO2 from actively metabolizing sites
How does CA-II change the rate of reaction?
CA-II increases the rate of hydrolysis by a factor of 10^7
What is the structure of CA-II
- the zinc atom lies near to the bottom of a 15 Å deep cleft.
- 3 His and one water coordinated
Give the mechanistic steps of CA-II
- Hydrophobic pocket next to Zn2+ captures CO2
- Nucleophilic attack on the carbon of CO2
- Conformational rearrangement at Zn centre
- Productdisplacementbysubstitutionwithwater
- His-64 as proton acceptor via a chain of hydrogen bonded water molecule (rds)
What is CPA?
Where is it found?
What does it catalyse?
- Carboxypeptidase A
- found in the pancreas
- helps assimilate ingested proteins
How is CPA selective?
- The cleavage of proteins only occurs at the C-terminal amino acid
- high selectivity for substrates in which the C-terminal amino acid contains a large aliphatic or Ph substituent.
Describe the structure of CPA
- CPA has a molecular weight of approx 34,500
- contains 307 amino acids and one zinc atom.
- Near the surface of the protein is a pocket containing the Zn2+ cation ligated by one bidentate Glu and two His residues.
- The 5-coordinate sphere is completed by a water molecule.
- Also vital to the mechanism are a guanidium ion of Arg-127 and the carboxylate group of Glu-270.
What is the proposed mechanism for CPA?
- Peptide is “manoeuvred” into position using hydrophobic and H-bonding interactions
- Arg-127 polarises the carbonyl group activating it towards nucleophilic attack by the Zn bound “activated” H2O
- Cleavage of the C-N bond facilitated by Glu-270
- Protontransferandpeptideandnewamino-acidleave
