Ch 9B Lecture
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Explain the cycle of carbon dioxide.
- in order to transport via RBC, you need bicarbonate
- Once in the lungs, a rapid conversion back so that CO2 can exit body
Carbonic anhydrases do this
WHat happens with carbonic anhydrase as pH increases?
the rate of the reaction increases. Histidine seems to be a candidate for this. But, it actually isnt. Itnstead, it is covalently bonded to a zinc atom in the active site, which interacts with H2O, which then attacks CO2
- less reactive; water does not dissociate a great deal; normal pKa of water arouund 15.
- Interaction of water with zinc--> loss of protn --> OH (strong nucleophile)
Zn formsa strong nucleophile, lowering the pKa of water
What is the short little mechanism for zinc bound water in carbonic anhydrases?
- zn forms a strong nucleophile by removing a H--> Co2 comes in and the hydroxide attacks the carbon dioxide
- There is no disruption of tertiary structre due to the presence of negative charge. Water will displace the HCO3-
What is key to the reaction of carbonic anhydrase?
lowering the pKa of water
Explain the reaction in terms of buffer.
In physiological conditions, the reaction must go rapdily in forward and reverse direction. In tissue, it must covert to bicarbonate. In lungs, it must be converted back to CO2. In the absence of buffer, the eq. constant is way different since the diffusion of the protons is a limiting factor. There will be a disparity between substrate and product
What does the buffer allow?
allow the f and r reactions to happen at similar rates which should take place for us to live
Why is buffer helpful?
because if we are producing protons at a rapid rate, those protons can't hang in the active site. They build up and something needs to get them out and hold them infor a while. So, buffers do this. Increasing buffer increases kcat
How can you speed up the reaction?
shuttle protons into and out of active site. Proton is immediately shuttled to the His residue, which is shuttled to the buffer that is present all around. It also gives up the H to g back
Convergent evolution regarding carbonic anhydrase?
zn is the convergent part
- key is the substrate: dsDNA
- major enemy of bacteria are viruses. Bac evolved ability to cleave foreign DNA and have a whole suit of enzymes that can chop them up prior to them harming them
What are the problems that must be overcome with restriction endonucleases?
- Host DNA of bacteria must not be cut
- double stranded DNA means that both strands must be cut
What do the enzymes do?
- they cut somewhere within the sequence.
- When they come apart, it results in sticky ends (small portions of single strands)
What must the enzyme overcome?
it must overcome the fact that the substrate is ds and must cleave both strands in close proximity to each other to make sure H bonds are overcome
a break in teh backbone of DNA is a nick. All cells have an enzyme called DNA ligase that goes through ligation. And, if a nick is present, it can be repaired.
What do restriction enzymes cleave?
the phosphodiester bond, causing a free 3'- oxygen end and a free 5'- phosphate end
How many mechanisms are there? Explain the first.
- nucleophile attacks and breaks a bond. Nucleophile is not activated since H is attached
- The intermediate is in the active site and covalently binds to substrate. One half of the substrate remains, but the other half does not.
- Water will attack the intermediate, hydrolyzing to produce the final products
Whyat is the second mechanis?
an activated water molecule attacks directly, hydrolyzing in one step
Why type of reaction is it? What does mechanism one result in? What does mechanism two result in?
in line displacement
- retention of stereoconfig
What does the TS of this reaction look like?
it is a pentacoordinate system
How do bacteria protect their own DNA?
they methylate the adenine nucleotide at the 5' end of hte reaction sequence. DNA methylases do this. The methyl group occupies enough space such that the substrate no longer fits prooperly in the active site.
How did they prove which mechanism it was?
they replaced one oxygen with sulfur and the incoming oxygen from water with oxygen-18. Water came in and resulted in inversion.
What do restriction endonucleases need?
divalent cations that are not involved in the reaction mechanism. Metal chelators can inhibit the enzyme by scavenging the ions.
What distinguishes cognate DNA from noncognate DNA?
though enzymes bind to both cog and non, cog DNA is the only kind of DNA that gets deformed. If there is a sequence in that DNA, it is able to form a kink between the T and A bases in the sequence (if EcoRV)
Amino acid side chains form __.
- very specific H bonds with DNA to hold it therem.
- Ben bound, DNA gets bent, foriming a kink.
The enzyme is instantly scanning DNA and can hold on tightly. But, it will not cleave until the sequence is found, at which point attractions hol dthe DNA in place. Interactions via bininding energy cause the kink to form, lowering Ea--> DNA cut
What is the deal with evolution and restriction endonucleases?
the same elements are located in allof the enzymes. They are key to the funciton of the enzyme. REcognition sequence is not hte same, however. Difference in specificity is due to the junk and structural elements that lie around the similar elements, as well as changes in one or two amino acids in the active.
Myosins do what?
- take energy from hydrogen or ATP so t can power thingks in our cell
- we capture energy associated with it by cleaving the bond. Myosins are the answer.
What is the structure
fibrous region and two globular ATPase domains within which is the enzyme activity that will break bonds and liberate energy
We use the same molecule as __.
THe shape was detected by __.
ATPase must have what?
The mechanism is the same as__
- slime molds
- divalent cation bound
- in line displacement
What is the function of Mg2+?
binds to ATP in a specific way so at so position the last phosphorusn near molecules that can hydrolyze it. In its absence, ATP cannot fix it
What happens when H2O is in?
activates H2O molecule, creating a nucleophile, no basic group but serine is in the active site and donates H to one O on phosphorus and then picks up H from the H2O. ATP is acting as own base to pick u a H and make its own nucleophile
Myosins can take small changes in shape and do what?
transform them to very large changes out of the periphery. Large change is equal to a step in the myosin
Difference in structure between ATP and ADP?
two different structural onformations that determine whether its ATP or ADP in the active site
What gets teh produt to leave the active site?
the conf changes that occur in the enzyme after hydrolysis is that forces molecule out
produts don't really leave the active site, they remain there. So, the conf changes encourage them to leave
What is the p loop
a loop that contains several glycine residues that are conserved between more closely related members ; it reacts with the phosphoryl gorups on the bound nucleotide
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