Exam 2 Flashcards
Carbonic Anhydrase
Carbonic Anhydrase
-Converts carbon dioxide to carbonic acid which partially dissociates to bicarbonate and proton
-obeys Michaelis menten kinetics
-Humans contain at least 7 carbonic anhydrase genes
-A buffer stimulates activity of carbonic Anhydrases
effect on pH:
-Maximal activity at higher pH
-Kcat/activity increases at approximately 7, there the group may have pka around 7 ( Group is Zinc not Histidine)
Carbonic Anhydrase II:
-Red Blood cells contain carbonic anhydrase II
Requires Zinc (Zn2+) which bonds to 4 Ligands
-3 to histidine (Imidazole Group)
-1 to water
3 families of Carbonic Anhydrase Alpha -Human enzymes-> Red Blood Cells Beta -Plants and Bacteria-> (Calvin cycle) Gamma -Arachea * Use of Zinc coordinated to 3 His and 1 Water has arisen 3 times-> CONVERGENT EVOLUTION
Nucleoside Monophosphate (NMP Kinases)
Nucleoside Monophosphate Kinase
Adenylate Kinase
Function
-Transfers a gamma phosphate from a nucleoside triphosphate to a nucleoside monophosphate producing a nucleoside disphosphate
Divalent cations (Mg2+, Mn2+, etc) required for enzymes using nucleotides as substrates
**not bound to enzyme active site, but bound to substrate (nucleotide)
-Substrate= ATP-Mg2+
Forms 6 coordination bonds (octahedral arrangement)
-two to oxygen
-4 to water
-bind to various oxygen in various combinations
-Numerous stereochemical orientations
-Increases interaction with enzyme=increased binding E
Xray crystal structures of enzymes are homologues
- Beta sheet surrounded by alpha helixes
- P-Loop
P-loop in adenylate Kinase
Phosphate Binding Site
-conserved Motife GXXXXGK binds ATP via Phosphate groups
-common to many nucleotide binding enzymes
P Loop mech:Binds to ATP-Mg2+
- Aspartic acid of adenylate kinase binds to ATP-Mg2+ complex through H-Bond to water, which induces a conformation change in adenylate Kinase (Induced Fit model)
- P loop closes over phosphates of ATP, especially B phosphate, and Gamma phosphate is now aligned next to NMP binding site
- Binding occurs at NMP binding site and causes additional conformational changes
- Catalysis by approximation
Other proteins contain P Loop NTPase Domain
- ATP synthase, Myosin, G protein, Tu (elongation factor),Helicases (DNA and RNA)
- P loops undergo conformational change upon NTP binding and hydrolysis
Restriction Endonuclease
Endonuclease Type II
Function
-cleave specific DNA base sequence (recognition site)
-Catalyzes Hydrolysis of Phosphodiester bonds
a)produce 5’ PO4 and 3’ OH
b) Mg2+ activates water, creating a nucleophile which attacks the phosphorus of the DNA
Found in Bacteria and Archaea
- require Mg2+ or other divalent cations
a) Mg2+ is bound by: 2 Asp or EcoR V, Phosphoryl Oxygen of DNA, and Water - 2 mechanisms
a) Both use pentacoordinated Intermediates
b) differ in number of displacements
Transition state
- Bipyramid Geometry-nucleophile attached to one apex; LG attached to the other apex
- Inverted Stereochemistry
Mech 1: 2 steps
1) formation of covalent intermediate
2) Hydrolysis to final product
- 2 inversions of stereochemistry
- RETAIN orientation of phosphorus
Mech 2: Direct Hydrolysis
- Single Inversion of stereochemistry
- Inverted configuration of Phosphorus
Experiment do differentiate between 2: Hard to determine orientation of Phosphorus so use Protocol
-Phosphorothioate Labeled DNA
-Water with O18
-determine by the orientation of water’s O18 relative to S
Results- Mech 2, because the orientation of water was inverted
Example of Restriction Endonuclease
EcoR V=Recognition Site 5’GATATC 3’
- cleaves methylated DNA
- protects-> methylation of 5’ adenine prevents H-bonding with Asn thus disrupting interaction between DNA and Enzyme
Interaction with Cognate DNA:
G:C BP H-bond with EcoR V
-backbone O and H-N of Gly (Diff Gly residues)
-backbone H-N of Asn
A:T BP H-bond with EcoR V
-backbone O and H-N of Asn (same Asn residue)
-r group Oxygen of Thr
X-ray structure of EcoR V
-cannot be determined due to cleaving of cognate DNA:Mg2+
Recognition Site Produces 2 fold rotational symmetry
- inverted repeats
- Restriction endonuclease binds equally with cognate DNA and non cognate DNA
Cognate DNA recognition site DISTORTS and produces additional interactions with EcoR V
- Free E (binding E) produces additional interactions with EcoR V
- Middle 5’ TA 3’ distorts and brings phosphate of DNA cleavage site into proximity of Mg binding site in active site of EcoR V. Mg binds completing catalytic apparatus:
Noncognate DNA Does NOT distort
- lack of distortion (in non cognate DNA) does not allow binding of magnesium, so catalytic apparatus not assembled
- lack of Mg2+, EcoR V binds equally well with cognate DNA and Noncognate DNA
Mechanisms of Binding:
NMP-Kinase vs Restriction Endonucleases
-diff in divalent cations in each
Restriction Endonuclease/EcoR V
Cognate DNA recognition site Distorts
-middle 5’ TA 3’ distorts and brings phosphate of DNA cleavage site into proximity of magnesium binding site in active site of EcoR V, and the magnesium binds completing the catalytic apparatus
NMP Kinase/Adenylate Kinase
P Loop of Adenylate Kinase binds to ATP-Mg2+ and induces a conformation change in adenylate kinase
-Aspartic acid of adenylate kinase binds to ATP-Mg2+ complex through hydrogen bond of water,
-which induces a conformational change and the P Loop lid closes over polyphosphates of ATP, and the gamma phosphate is now aligned with NMP binding site, and binds causing additional conformation changes
-catalysis by approximation
**BOTH REQUIRE DIVALENT CATION only difference is what it binds (Mg2+)
NMP kinase Mg binds to substrate (Nucleotide-ATP)
Restriction Endonuclease Mg binds to Cognate DNA then that binds to EcoR V
Restriction Endonuclease Magnesium bound by: -two aspartate (asp) residues of EcoR V -phospohryl oxygens atoms of Cognate DNA -water
NMP Kinase-
Magnesium forms 6 coordinated bonds (octahedral arrangement)
-2 to oxygen
-rest to water (4)
Proteases
Spontaneously Cleave peptide bonds (-Gibbs Free Energy) of proteins by hydrolysis (addition of water
Types of proteases: -serine protease Ex:Chymotrypsin a) Nucleophile-Serine 1st (2nd-water) b) Histidine activates serine c) Asp stabilizes His -Cysteine Protease a) Cysteine-1st nucleophile (2nd-water) b) His activates cysteine -Aspartyl proteases A)2 aspartic acids activate water creating a nucleophile (1st and 2nd) -Metalloproteases active site a)bound metal (zinc) activates water molecule creating nucleophile (1st and 2nd)
Common Features:
- activate water or other nucleophiles
- polarize the peptide carbonyl group
- stablize the tetrahedral intermediate
Protease Inhibitors as Drugs
Indinavir-inhibits HIV protease
Captopril
- inhibits angiotensin converting enzyme (ACE)
- regulates BP
Subtilisin
Nonhomologue of chymotrypsin
- uses catalytic triad
- bacteria, archaea, eukwrya
- lack 1’, 2’, 3’ similarity to chymotrypsin
Carboxypeptidase II
Nonhomologue of Chymotrypsin
- uses catalytic triad
- Wheat
- Lack 1’, 2’, 3’ similarity to chymotrypsin
Trypsine
Serine Protease
- contain catalytic triad
- activates more trypsin and other zymogens (chymotrypsinogen)
Binding pocket adds specificity
- (hydrolyzes)cleaves peptide bonds after large positively charge side chains (R,K)
- contains Asp
Elastase
Serine Protease
-contain catalytic triad
Binding pocket adds specificity
- cleaves peptide bonds after small side chain amino acids (A, S)
- contains Valine
Chymotrypsin
Serine Protease
Function:
-hydrolyzes protein in small intestine
-cleave peptide bonds carboxyl end of Large hydrophobic amino acids (M) and Aromatic Amino Acids (M)
-Binding pocket. adds specificity (homologues Chymotrypsin)- contain large hydrophobic amino acids (W, M, G)
-Catalytic Triad: Ser-His-Asp
Structure:
- 3 subunits bonded by 4 Disulfide bridges
a) 2 intrachain
b) 2 interchain
Mechanism: 2 steps
1) acylation
2) deacylation
synthesized from 245 amino acid Zymogene (precursor) in the pancreas (acinar cells)
-Trypsinogen
Activated by proteolytic cleavage in cascade
Identification of catalytically active Serine:
- Chymotrypsin contains 28 serines
- use reagent DIPF (diisopropylphosphofluoridate)-only one serine reacts with
Artificial Chromogenic Substrate
- n-acetyl-L-phenylalanine p-nitrophenyl ester
- produces Yellow product
a) p-nitrophenolate - Substrate contains ester bond instead of amide bond found in proteins
Kinetics:
- Obeys Michaelis Menten Kinetics
- Reaction monitored by Stop flow method (Rxn is 2 steps)
a) Rapid Burst
b) Slow steady state reaction
ATCase
Aspartate Transcarbamoylase
1) Allosteric Regulation-first committed step in the multi step pathway to synthesize pyrimidines
2) Multisubunit Enzyme 2C3 + 3R2->C6R6
2 catalytic subunits
-c3=trimer
a) subunits stack on top of one another
b) each subunit contains 3 active sites
-Purified c subunits exhibit catalytic activity and unresponsive to CTP
3 Regulatory subunits
-r2=dimer
a)each subunit contains 2 binding sites to CTP (allosteric inhibitor); stabilizes T form
b) contains 2 Zn2+ binding sites
Each C subunit connects to 2 R subunits through a structural domain in the R subunits that is stablilized by Zn2+ ion bound to 4 cysteine bonds
3)Active site- formed between to catalytic trimers
-one-> 2 Thr and Arg, 1 His and Gln
adjacent-> Ser and Lys
4) 2 existing state: alternate between T form and R Form
-T form
a) low affinity for substrate
b) CTP binds to inhibit E activity
-R form
a) high affinity for substrate (cooperativity)
At equilibrium with no substrate or CTP; 200 T: 1 Rform
5) CTP binds with a domain of regulatory subunit not in contact with catalytic subunit and inhibits E activity by stabilizing T form
6) Substrate binds and conversion from T to R form
- substrate= carbamoyl Phosphate/aspartate or PALA
Pala-Bianalog Substrate
- similar reaction intermediate
- competitive inhibitor to ATCase, binds to active site
7) exhibits concerted model of cooperativity while other cooperativity exhibit a mixture of concerted and sequential models
Sucrose Gel Centrifugation of ATCase
Sucrose Gel Centrifugation
-seperates by difference in size
1) use p-hydroxymercuribenzoate which reacts with sulfhydryl of cysteine resulting in the breaking of the Zn2+ and cysteine bond allowing the R and C subunits to dissociate without Zn2+ attached
2) Have to get rid of p-hydroxyemercuribenzoate from subunits thus us mercaptoethanol which produces pure R and C subunits which restores activity
- can now study individually
Two Model of Cooperativity
Concerted Model
-binding of one substrate leads to “all or none” transition to R state and all active sites transitions to R state
Sequential Model
-binding of one substrate leads to transition to R site of neighboring active sites but not all active sites
