浙江大学生物无机化学课件Chapter 5

3. Ready formation of low coordinate binding sites (> acidity)
Zinc Metalloenzymes
The unique feature of all of these enzymes is the presence of an activated water molecule bound to Zn(II). The pKa of metal-free water is 15.7 but can be reduced to 10 in [Zn(H2O)6]2+ and 7 with three Ndonors. This allows for facile ionization of H2O.
OH2 Zn HN N N N H N NH
The active site lies near the bottom of a 15 Å deep cleft.
Carbonic Anhydrase II
Active site
Carbonic Anhydrase II
An ordered array of water molecules in the active site is of special importance Rate determining step is NOT the CO2 - HCO3conversion but rather proton shuttling under participation of amino-acid side chains and water network
Carbonic Anhydrase II
Description:
Consists of 260 amino acid residues plus one Zn2+ ion.
A zinc ion coordinated by three N atoms (histidines) with the fourth site occupied by a H2O molecule.
Enzymes
Oxidoreductases
Oxidases, Fe, Cu Nitrogenases, Fe, Mo, V Hydrogenases, Fe, Ni Reductases, Fe, Cu, Mo Hydroxylases, Fe, Cu, Mo Superoxide dismutases, Fe, Cu, Mn
kD single polypeptide sub-units, each of which contains two zinc ions. One Zn atom is in the catalytic site which also binds NAD+. The other Zn atom plays a structural role binding four cysteinate residues. In the oxidation of alcohol, two hydrogen atoms are removed - one to the 4-position of NAD+ and the other as a proton. The transfer to NAD+ is generally thought to be a hydride transfer.
Carbonic Anhydrase II (CAII) is present in red blood cells and catalyzes the reversible hydration of CO2.
H2O + CO2 [HCO3]- + H+
This process is slow (k = 0.037 s-1 ) but is essential for removing CO2 form actively metabolizing sites. CAII increases the rate of hydrolysis by a factor of 107!!!!! Can also catalyze hydrolysis of esters and aldehydes
Transport & Storage
Electrons
Siderophores, Fe Skeletal, Ca, Si, Transfer. K, Na
Photoredox
Photons
Chlorphyll, Mg Photosystem II, Mn
Metals
Ferritin, Fe Transferrin, Fe Ceruloplasm, Cu
Hydrolases
Phosphatases, Mg, Zn, Cu Aminopeptidases, Mg, Zn Carboxypeptidases, Zn
Synthetases
Vitamin B12 coenzyme, Co
Zinc Metalloenzymes
Zinc is the 2nd most abundant trace element in humans and is required for > 100 enzymes.
OH2 N N N carbonic anhydrase II Zn OH2 N S S alcohol dehydrogenase Zn OH2 O Zn N O N carboxypeptidase A 羧肽酶
Zinc Metalloenzymes – Reactions Catalyzed
condensation R XH + HO A hydrolysis R X A + H2O
X-ray Structures of Active Sites in Carboxypeptidase A and Carboxypeptidase G2
Alkaline Phosphatase(碱性磷酸酶）
Asp327 His331 Asp51
HN O N Zn N
His412
O
O
O
O O Zn N
R
R N
HO
N
O NH2
烟酰胺腺嘌呤二核苷酸NAD
Nicotinamide Adenine Dinucelotide
NH2 H
NADH
H
Hydride transfer
Alcohol dehydrogenase
Liver alcohol dehydrogenase comprises two 40-
Carboxypeptidase A(羧肽酶)
Carboxypeptidase A (CPA) is a pancreatic enzyme胰腺酶 that cleaves the carboxyl terminal amino acid from a peptide chain by hydrolyzing the amide linkage. There is a high selectivity for substrates with large terminal aliphatic or phenyl substituents
e.g X = NH A = O
R'
peptidases, lactamases, collagenases, dehydratases
e.g X = O A = O e.g X = O A = PO32-
R'
esterases
phosphatases
Carbonic Anhydrase II
(碳酸酐酶 )
Asp369
NH
His370
HO N H H HN
Arg166
H NH
Ser102
NH
Zinc polarises the substrate making it a better electrophile Asp-51 also coordinated to magnesium cation
Asp327 His331 Asp51
Phosphatase
Alcohol dehydrogenase
(醇脱氢酶)
Alcohol dehydrogenase converts alcohols to aldehydes or ketones
Alcohol dehydrogenase
R2CHOH + NAD+
co-enzyme
R2CO + NADH + H+
His412
O
O
O
O O Zn
Asp369
HN O N Zn O
O
O
O O Zn
Asp369
O O H O H HN
Arg166
N P O O H NH
NH
His370
N
His412
O P H O H HN
Arg166
O HO O H NH
N
NH
His370
N H
Ser102
N H
Ser102
NH
NH
MW of ca. 34,600 Roughly egg-shaped with approx. dimensions 50 Å 38 Å x
Active Site

A cleft on one side contains the Zn2+ ion = active site The zinc ion is coordinated by two N atoms (histidines) and two O atoms (glutamate) in the protein chain; the fifth site is contains H2O.
Carboxypeptidase A
Active site
OH2 Zn N O O NH
HN
N
Histidine-196 Glutamate-72
Histidine-69
Bond that will ultimately be cleaved
R’ is accommodated in a hydrophobic pocket
HN O N Zn N
His412
O
O
O
O O Zn N
Asp369
NH
His370
O O R O H HN
Arg166
P
-O O H NH
Ser102
N H
H2O -ROH
ROPO32- HOPO32-
NH
Asp327 His331 Asp51
Asp327 His331 Asp51
HN O N Zn N
Important chemical features
1. Is as a strong Lewis acid. 2. NOT redox active. 4. Accessible coordination numbers of 4, 5, 6. 5. Easily deformed coordination geometry. 6. Easily undergoes mono-ligand substitution chemistry
H2 …Pro-Leu-Glu-Phe ...Pro-Leu-Glu + Phe O carboxypeptidase A
Carboxypeptidase A
Basic Description:

Consists of 307 amino acid residues plus one Zn2+ ion
•The catalytic zinc is bound to Cys-46, Cys174 and His-67. An ionizable water molecule occupies the fourth position on the Zn. •The water is H-bonded to the hydroxyl of Ser48. The oxygen atom of the substrate is believed to bind directly to the zinc ion. The nicotinamide ring of NAD+ is bound close to the zinc.
第5章 金属蛋白和金属酶
Chapter 5 Metal Proteins and Metal Enzymes
Metallo-Biomolecules
PROTEIBiblioteka BaiduS NON-PROTEINS
Transport & Storage
Electrons
Cytochromes, Fe Iron-Sulfur, Fe Copper blues, Cu Oxygen Myoglobin, Fe Hemoglobin, Fe Hemerythrin, Fe Hemocyanin, Cu
Dipolar interaction H-bonding
H-bonding
Nucleophilic attack on carbonyl C-atom
Ionization
Cleavage of the C-N bond
Proton transfer to give NH3+ & COO- groups