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__ is the major end product of aerobic metabolism. It is __, __, and, while in the RBCs, __, to form a strong acid, __, which is converted into __ on the loss of a proton
This proceeds fast even without a __
- Carbon dioxide
- released into the blood
- transported to the lungs for exhalation
- carbon dioxide reacts with water
- carbonic acid
- bicarbonate ion
Carbon dioxide hydration and HCO3-
dehydration are often coupled to rapid processes. Thus, almost all organisms contain enzymes, referred to as __, that __
- carbonic anhydrases
- increase the rate of reaction beyond the already reasonable spontaneous rate.
Ex: Carbonic anhydrases do what?
Conversely, they do what?
Furthermore, both __ and __ are substrates and products for a variety of enzymes, and the rapid interconversion of these species may be necessary to ensure appropriate substrate levels
dehydrate HCO3- in the blood to form CO2 for exhaling as the blood passes through the lungs
convert CO2 into HCO3- to generate the aqueous humor of the eye and other secretions
CO2 and HCO3-
Carbonic anhydrases accelerate __dramatically
Carbonic anhydrase has a bound __ion, which is necessary for catalytic activity; and, today, more than 1/3 of all enzymes either contain __ or require the addition of such ions for activity
- CO2 hydration
- bound metal ions
Metal ions have several properties that increase chemical reactivity: __(3)__
There are about __carbonic anhydrases; and __ has been the most studied
- their positive charges, their ability to form strong yet kinetically labile bonds, and, in some cases, their capacity to be stable in more than one oxidation state
- carbonic anhydrase II
__is found only in the __ state in biological systems; and it is essentially always bound to __; in carbonic anhydrase, __ are occupied by the __ of __and an additional __ is occupied by a water molecule. Because the molecules occupying the coordination sites are __, the overall charge on the Zn(His)3
unit remains +2
- four or more ligans
- three coordination sites
- imidazole rings of three histidine residues coordination site
· How does the zinc complex facilitate carbon dioxide hydration?
o At pH __, the reaction proceeds near its __. As the pH __, the rate of the reaction drops
The midpoint is near pH 7, suggesting what?
The __ form participates more effectively in catalysis
- a proton lost plays an important role int eh activity of carbonic anhydrase
A variety of evidence suggests that, although some amino acids like histidine have pKa values near 7, the group responsible for this transition is the __, which causes a __
With a lower pKa, many water molecules lose a proton at __, generating a substantial concentration of __
- zinc bound water molecule
- reduction from pKa of 15.7 to 7
- neutral pH
- hydroxide ion
A __is a potent nucleophile able to attack __ much more readily than water does. Adjacent to the zinc site, carbonic anhydrase also possesses a __ that serves as a __ for __
- zinc bound hydroxide ion
- carbon dioxide
- hydrophobic patch
- binding site for carbon dioxide
From this info, a simple mechanism for carbon dioxide hydration can be proposed: Steps one and two?
- § The zinc ion facilitates the release of a proton from a water molecule, which generates a hydroxide ion
- § The carbon dioxide substrate binds to the enzyme’s active site and is positioned to react with the hydroxide ion
From this info, a simple mechanism for carbon dioxide hydration can be proposed: Steps three and four?
- § The hydroxide ion attacks the carbon dioxide, converting it into bicarbonate ion
- § The catalytic site is regenerated with the release of HCO3 and the binding of another molecule of water
Thus, the binding of a water molecule to the zinc ion favors the __ by facilitating __and by __
This has been tested by the __, where a synthetic ligand binds zinc through __. One water molecule remains __ int eh complex and direct reasurements are taken, revealing that this water molecule has a pKa value of 8.7, not as low as the value for the water molecule in carbonic anhydrase but lower than the value for free water.
- formation of the transition state
- proton release
- positioning the water molecule to be in close proximity to the other reactant
- synthetic analog model system
- four nitrogen atoms
- bound to the zinc ion
At 9.2, this complex does what?
Although its rate of catalysis is much less efficient than catalysis by __, the model system strongly suggests that the __
- accelerates the hydration of carbon dioxide more than 100-fold
- carbonic anhydrase
zinc-bound hydroxide mechanism is likely o be correct
In the first step of the carbon dioxide hydration reaction, what must happen?
The reverse reaction’s rate is limited by the __, which diffuse very rapidly. Thus, the backward rate constant k-1
must be less than 1011
- proton shuttle
- rapid regeneration of the active form of the enzyme
the zinc bound water molecule must lose a proton to regenerate the active form of the enzyme
rate of proton diffusion
Because equilibrium constant K is equal to k1/k-1, the forward rate constant is given by k1=K*k-1
In other words, the rate of proton diffusion does what?
However, if carbon dioxide is hydrated at a rate of 106
s-1, then every step in the mechanism must take place at least this fast
limits the rate of proton release to less than 104s-1 for a group with pKa=7.
The highest rates of carbon dioxide hydration require the __, suggesting that the buffer components participate in the reaction
The buffer can __ or __; and the advantage is that, whereas the concentrations of protons and hydroxide ions are limited to 10-7
M at neutral pH, the concentration of buffer components can be __, of the order of several millimolar
- presence of buffer
- bind or release protons
- much higher
The molecular components of many buffers are what?
; and so, carbonic anhydrase II evolved a __ to allow buffer components to participate in the reaction from solution. The primary component of this __ is __.
too large to reach the active site of carbonic anhydrase
shuttle is histidine 64
This histidine does what?
Thus, catalytic function has been enhanced through the evolution of an apparatus for doing what?
Because protons participate in many biochemical reactions, what
is crucial to the function of many enzymes and what is explained?
transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer
controlling proton transfer from and to the active site
the manipulation of the proton inventory within active sites
explains the prominence of acid-base catalysis
__ has generated zinc-based active sites in different carbonic anhydrases
o Besides __, two other families have been discovered.
They contain zinc but are not similar in sequence to the __.
- Convergent evolution
- alpha-carbonic anhydrases x2
The __ are found in higher plants and in many bacterial species.
Explain where the zinc is. Moreover, the overall enzyme structures are unrelated to those of the alpha-carbonic anhydrases
In plants, these enzymes facilitate the __
the zinc is bound by one histidine residue and two cysteine residues.
accumulation of carbon dioxide
The __ have a crystal structure with three zinc sites extremely similar to the zinc site in the alpha-carbonic anhydrases. In this case, howver, what is different?
The very striking __ present in this enzyme is different from any structure present in the alpha and beta versions. Thus, __ has generated __ that rely on coordinated zinc ions at least three times
- gamma-carbonic anhydrases
- the three zinc sites lie at the interfaces between the three subunits of a trimeric enzyme
- left-handed beta-helical structure
- convergent evolution
- carbonic anhydrases