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Flashcards in Chapter 9 Deck (70):

what biochemical challenge do serine proteases overcome? what is an example?

must promote reactions that are infinately slow at neutral pH. chymotrypsin.


what biochemical challenge do carbonic anyhdrases overcome?

must achieve a high rate of reaction to keep up with physiological processes


what biochemical challenge do restriction endonucleases overcome?

must achieve exquisite specificity for DNA sequences.


what biochemical challenge do nucleoside monophosphate NMP kinases overcome?

must transfer a phosphoryl group from ATP to a nucleotide instead of water.


what are the 4 catalytic strategies often employed by enzymes?

1. covalent catalysis
2. general acid-base catalysis
3. metal ion catalysis
4. catalysis by approximation


what is covalent catalysis

the active site contains a reactive group, usually a nucleophile, that becomes temporarily covalently attached to the substrate during catalysis


what is general acid-base catalysis? what is an example?

a molecule other than H2O plays the role of a proton donor or acceptor (chymotrypsin uses a histidine as a base catalyst to enhance the nucleophilic power of serine)


what is metal ion catalysis?.

1. metal ions can serve as an electrophile and stabilize a negative charge of a reaction intermediate (magnesium ions in EcoRV)
2. can generate nucleophile by increasing acidity of a nearby molecule such as H2O in the hydration of CO2 by carbonic anhydrase
3. can bind to substrate, increasing number of interactions with the enzyme


what is catalysis by approximation?

bringing 2 substrates together along a single binding surface on an enzyme. for example NMP kinases bringing 2 nucleotides together


preteases must be broken down because:

1. protein ingested must be broken down into amino acids to be absorbed, proteases can regulate other proteins through proteolytic cleavage. proteins that have served prupose must be recycled


how do proteases cleave proteins?

hydrolysis reaction


why are proteases needed if the hydrolysis reaction is thermodynamically favored?

hydrolysis reaction is too slow


what is chymotrypsis?

serine protease that cleaves proteins on carboxy side of aromatic or large hydrophobic amino acid


why are peptide bonds so stable? how does chymotrypsin break it?

due to resonance. they have a partion double bond character. Carbonyl C less electrophilic and less susceptible to nucleophilic attack. enzyme must generate powerful nucleophile to cleave bond


what type of catalysis does chymotrypsin use?

covalent catalysis


how is kinetics of an enzyme monitored?

by having an enzyme act on a substrate that will form a colored product.


describe kinetics of chymotrypsin catalysis

2 stage reaction: initial rapid burst phase followed by slower formation to reach steady state.


what explains this 2 stage reaction?

initial formation of a covalent enzyme-substrate intermediate called acyl-enzyme intermediate. followed b intermediate is hydrolyzed to release the carboxylic acid component of the substrate and regenerate free enzyme


what is chymotrypsin's structure?

it is spherical and consists of 3 chains lined together by disulfide bonds ( 2 interstrand and 2 intrastrand). it is intially snythesized as a single chain which undergoes cleavage and activation to form 3 chains


what is the catalyitc triad of chymotrypsin and how does it work?

three residues (asp, his, ser) interact. histidine positions and polarizes serine sidechain so that it can be deprotonated. histidine then accepts the proton from serine. aspartate residues orient histidine to make it a better proton acceptor through hydrogen bonding and electrostatic effects. serine is converted to a potent nucleophile an alkoxide ion


how is the oxyanion stablizied?

hydrogen bonds linked NH groups and negatively charged oxygen to stabilize the tetra hedral intermediate


why does chymotrypsin prefer to cleave after large hydrophobic groups?

there is a deep hydrophobic pocket into which the long uncharged side chains of residues can fit, specificity dpends on the identity of the amino acid on the N-terminal side of the cleavage site.


how would you increase specificity of a protease?

have multiple specificty pockets.


what are differences in specificity due to?

small stuctural differneces.


how is specificity for trypsin and elastatse determined?

1. trypsin cleaves afer residues with long positively charged side chains (aspartate residue in pocket attracts and stabilizes substrate)

2. - elastase cleaves after amino acids with small side chians such as alanine and serine; bulky valine residues close off mouth of pocket so that only small amino acids can enter


what proteins have similar catalytic triad and oxyanion hole to chymotrypsin despite not being evolutionary related? what kind of evolution does this signifiy?

subtilisin and carboxypeptidase. convergent molecular evolution


what are other classes of proteases?

1. cysteine proteases

2. aspartyl proteases

3. Metalloproteases


mechanism of cysteine proteases

-cysteine residue activated by histidine engages in nucleophlic attack on the peptide bond


mechanism of aspartyl proteases

a pair of aspartic acid residues act together to allow a water molecule to attack the peptide bond. one deprotonated aspartate activates the attacking water molecule, the other protonated aspartic acid residue polarizes the peptide carbonyl group so that it is more susceptible to attack


mechanism of metal ion proteases

- active site contains a bound metal ion, usually zinc that activates a water molecule to attack the peptide carbonyl group


what is HIV protease?

retroviral aspartyl protease necessary for life-cycle of HIV. it cleaves newly synthesized polyproteins at appropriate places to create the mature protein components of an infectious HIV Virion. it is a dimer with each subunit contributing one aspartic acid residue


what are the common functions of active sites of proteases

1. activate a water molecule or other nucleophile
2. polarize the peptide carbonyl group
3. stabilize the tetrahedral intermediate


how does captopril treat high blood pressure?

inhibits angiotensin converting enzyme ACE which is a metalloprotease


how does indinavir treat HIV?

Inhibits HIV protease because it resembles the peptide substrate of HIV protease. has functional groups that bind to four specificity pockets of HIV protease. the active site covered by 2 flaps that fold over bound inhibitor, the OH of the central alcophol group interacts with the active site aspartate residues.


what does carbonic anhydrase do?

greatly increase speed of conversion of co2 + h2o bicarbonate


how many carbonic anhydrases in humans



which carbonic anhydrase in humans is well studied

human carbonic anhydrase II


what was the first known zinc containing enzyme

human carbonic anhydrase II


what proportion of enzymes contain metal ions?

more than 1/3


what properties do metal ions utilize to assist catalysis?

1. postive charge,
2. form relatively strong but kinetically labile bonds
3. in some cases, have the capacity to be stable in more than one oxidation state


describe zinc site in human carbonic anhydrase II

1. zinc ion is bound to imidazole rings of three histidines as well as a water molecule.

2. zinc site is located in a cleft near the center of the enzyme

3. zinc is always in the +2 state in biological systems

4. the protonation state of the water molecule is pH- dependent


how does pH affect carbonic acid? what pH does it transition at? what molecule is important for this?

transitions near pH 7.0 due to zinc-bound water molecule


what happens when water molecule binds to a positively charged zinc

reduces pKa of water molecule from 15.7 to 7


what are the effects of the lowered pKa of water?

water loses a proton at pH 7 which generates a lot of hydroxide ion bound to zinc ion. zinc bound hydroxide is a very good nucleophile that can readily attack carbon dioxide


what is the overall mechanism steps 1-4 of carbonic anhydrase?

1. zinc ion generates hydroxide ion
2. co2 binds to enzyme and is positioned to react with the hydroxide ion
3. hydroxide ion attacks the CO2 and converts it into bicarbonate ion
4. the active site is regenerated with the release of bicarbonate and the binding of another water molecule


what is the rate of carbon dioxide hydration limited by. how does the rate change?

it is 10^6/s but it is limited to 10^4/s by the rate of proton diffusion. water must be deprotonated before catalysis can occur


what affects the rate of carbon dioxide hydration

carbon dioxide hydration increases directly with the concentration of buffer. allows carbonic anhydrase to achieve high catalytic rates


what is a proton shuttle?

it allows buffer components to participate in the reaction despite being too large to access the active site


how does the proton shuttle work

histidine residue of transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer.


how does proton shuttle affect catalytic function?

it enhances catalytic function


what do most enzymes acting on nucleic acids require for activity? where are they found and what do they do?

require Mg2+ ions. they are located in active site and coordinated by aspartate residues and to posphoryl group oxygen atoms at cleavage site. one of the metal ions binds the water molecule that engages in nucleophilic attack


what are the recognition sequences for most restriction enzymes?

inverted repeats


what kind of symmetry do restriction enzymes have?

2- fold rotational symmetry


how does EcoRV cut dna?

2 loops from within enzyme (one from each subunit) directly hydrogen bond to specific base pairs. The central TA secquence of GATATC does not bind to the enzyme and is kinked. since the path of the DNA helical axis is distorted when binding to the enzyme, this facilitates breakage of the phosphate bond.


how does a restriction enzyme differ when it binds to noncognate DNA fragments?

noncognate DNA is not substantially distorted, lack of distortion has negative effects upon catalysis. There is no phosphate group to complete binding site for magnesium ions


how does methylation block cleavage

prevents formation of a hydrogen bond between the amino group and the side-chain carbonyl of asn 15. this disrupts other interactions between enzyme and dna so no distrotion of dna takes place


does type II restriction enzyme have a conserved fuction



NMK kinases (nucleoside monophosphate) does what?

transfers a terminal phosphate from NTP (usually ATP) to the phosphate group on a nucleoside monophosphate


what is the challenge for NMP kinase to promote?

this transfer without promoting the competing reaction of trfansfering the phosphate group from ATP to water (NTP hydrolysis)


are there many NMP kinases?

yes, NMP kinases form a family of homologus proteins


what is conserved in all NMP kinases

NTP binding domain is present consisting of a central beta sheet surrounded on both sides with alpha helices


what is an important structrual feature of NMP kinases?

a loop between beta sheet and first alpha helix-- called the P loop because it interacts with phosphate groups on the NTP (found in many important nucelotide binding proteins)


how does the p loop interact with atp

hydrogen bonds link atp to peptide NH groups and a lysine residue


what ion does NMP kinase need? how is this ion used?

Mg2+ (however not part of active site). NTPs bind these metal ions and this ATP-Mg2+ complex is the true substrate for the enzyme. interactions between Mg2+ and phosphoryl groups holds the nucelotide in a well-defined coformation that can be recognized by the eznyme


what are magnesium ions coordinated to?

coordinated to 6 groups in an octahedral arrangement (many structural isomers)
- usually 2 oxygen atoms are directly coordinated to the magnesium ion with other positions occupied by water molecules


what does the water bound to magnesium do?

water molecules interact with groups on the enzyme including conserved what aspartate residue.

!!! The magnesium ion provides additional points of interaction between
the ATP-Mg2+ complex and the enzyme


what are mg2+ 3 essential functions?

1. holds nucleotide in well defined conformations that can be specifically bound to the enzyme

2. neutralize polyphosphate charges, reducing nonspecific ionic interactions

3. provide additional points of interaction with the enzyme, either directly to the Mg2+ ions or indirectly with the Mg2+ ions through H bonds to the coordinated H2O molecules.


what changes occur when ATP Mg2+ binds to NMP kinase

P-Loop closes down on (beta) polyphosphate chain. to domain moves down to form lid over bound nucleotide. its gamma phosphoryl group is positioned for 2nd substrate NMP to bind (causes more conformational changes)


how does this catalytically competent conformation (that occurs when ATP-mg2+ binds to NMP kinase) prevent transfer of phosphoryl group to water?

enzyme stablizes the transition state that leads the transfer of phosphoryl group from ATP to NMP.
- both sets of changes only occur when both the donor and acceptor are bound.


what kind of catalysis does nmp kinase do?

catalysis by approximation