Chemistry 310 - Enzyme Mechanisms - Prof. Bryan Hanson

A List Summarizing Some Important Things to Keep in Mind

I will add to this list as we go along. You may wish to consult this page before each exam to remind yourself of the big picture. The date of last update is at the bottom of the page.

How do we know/determine a mechanism? What kinds of evidence can be used to deduce a mechanism?

Kinetics: the rate law tells us the species colliding in the rate determining step.
Stereochemistry: inversion of chirality vs retention vs racemization.
Site-directed mutagenesis: changing a particular amino acid at the active site can tell you if it is involved in the mechanism. A typical change is to take an acidic or basic side chain and mutate it to something small and non-polar. If this affects the reaction rate (enzyme activity), that amino acid was contributing to the reaction somehow.
Use of inhibitors. A properly designed inhibitor will bind to the active site but not react. Comparison of the inhibitor structure to the normal substrate structure can imply what's going on with a mechanism.
pH-rate profiles.

What the active site does

The active site supplies the amino acid side chains necessary for binding and carrying out the reaction (catalysis). Some amino acids may play both roles.
The active site organizes the reaction by putting all the players in close proximity, including but not limited to folding the substrate appropriately and arranging any metal ions or water molecules where needed.
The active site provides amino acid side chains that are complementary to the substrate in that they provide appropriate intermolecular forces for binding. The main intermolecular forces in decreasing strength are:
- electrostatic attractions (ion-ion attractions)
- hydrogen bonding (dipole-dipole attractions)
- cation-pi attractions (a unique force in which the orbitals of an ion interact with aromatic pi MOs)
- London forces (induced dipole-induced dipole attractions; you may know them as van der Waals forces)
Size-wise, a substrate must fit into the active site. It may be a little small, but never too large.

Reaction mechanisms we have learned

additions: two species are added across a pi bond (either an alkene or carbonyl group)
- Ad_E: electrophilic addition to an alkene; the electrophile is usually H+ or a carbonium ion
- Ad_NuC=O: nucleophilic addition to a carbonyl in which a nucleophile attacks the carbon of an aldehyde or ketone
- 1,4 additions, also called Michael additions or conjugate additions: Related to Ad_NuC=O except that there is an intervening double bond that acts to relay electrons into the carbonyl group. The intermediate formed is an enolate, which must be protonated to complete the reaction.
substitution: one group is substituted for another
- S_N1: nucleophilic substitution at saturated carbon, proceeding through a carbonium ion
- S_N2: nucleophilic substitution at saturated carbon, a single step "concerted" reaction
- S_NuC=O: nucleophilic substitution on a carbonyl group (characteristic of functional groups related to the carboxylic acid, which have a good leaving group on the carbonyl carbon).
Elimination: loss of (usually) a proton and a leaving group to give an alkene
- E1: same first step as S_N1, proceeds through carbonium ion
- E2: a single step reaction in which H and LG are simultaneously extracted by a base
- E1CB: CB = conjugate base. A particularly stable conjugate base is formed, which then pushes out a leaving group.

Reaction categories we have learned

This list complements the one above; for instance, one can talk about substitution rxns as a category of reactions. What is given here are terms that are frequently encountered. Every category or term here is an example of a particular mechanism listed above.

alkylation - typically occur via an S_N2 mechanism, they transfer alkyl groups (think SAM as an alkylating agent)
redox rxns (oxidation/reduction) - conversion of a carbonyl to an alcohol is reduction, the reverse is an oxidation. NADH is a reducing agent. Conversion of a carboxylic acid or a relative to a aldehyde is also a reduction.
hydration - addition of a water molecule across an alkene via A_dE
hydrolysis - "breaking with water", for example proteases and esterases, which operate via S_NuC=O.
condensation - condensing two carbon building blocks into a larger piece - aldol condensation, and Claisen condensation.
- the aldol condensation creates a β-hydroxy aldehyde or ketone
  - the product of an aldol condensation often eliminates to form an α, β-unsaturated aldehyde or ketone
- the Claisen condensation creates a β-keto ester, or any β-keto derivative of a carboxylic acid relative
- in both of these reactions it is the bond between the α-carbon and β-carbon of the product that is formed in the reaction.
acylation - the addition of an acyl group to a nucleophile. These occur via S_NuC=O. From the perspective of the C=O, it has been substituted. From the perspective of the incoming nucleophile, it has been acylated.
tautomerization - a 1,3 shift of hydrogen from carbon to N or O, with a corresponding shift in the pi electrons. Phenol is an example where the enol form is most stable; in most cases it is the keto form that is most stable.
decarboxylation - loss of CO₂, which can occur in several mechanisms/situations.

Cofactors & other important molecules & what they do

SAM: S-adenosylmethionine. Supplies a methyl group to a nucleophile via an S_N2 mechanism. Acts as an alkylating agent.
NADH/NAD+ and NADPH/NADP+. These are biological oxidizing and reducing agents. They act as though they deliver a hydride ion (H:–) to a carbonyl to reduce it, or receive a hydride in an oxidation. No free hydride exists, this is just how they appear to behave. The one that is used depends upon the enzyme and upon it's reduction potential.
CoASH: Coenzyme A, the business end is a thiol or sulfhydryl group, –SH, which serves carry acyl groups in a variety of reactions. When acylated, CoASH becomes a thioester, the most often mentioned example being AcCoA or acetyl CoA (CH₃COSCoA).
Enolates: the conjugate base of carbonyl groups where the alpha carbon has been deprotonated, and the charge is resonance stabilized. They are nucleophilic at the alpha carbon. They are also the conjugate bases of enols.
Biotin: a carrier of CO₂.
Thiamine pyrophosphate (TPP): A carrier of two carbon pieces, one of which is a ketone, the other can vary (there are a few exceptions to this notion). For example: –CO–CH₃ or –CO–CH₂OH. The active form is an ylide, a strange sort of creature that has a + and – charge adjacent, with the + charge residing on and N, P or S (the latter two are not seen in biochemistry AFAIK).
Lipoamide: contains a disulfide bond that gets oxidized and reduced at various points. Used to make a thioester of the dihydroform which then can be transthioesterified to a different thioester (e.g. of CoA). Lipoamide has to be regenerated by FAD which in turn is regenerated by NAD+.
PLP and PMP: these are the cofactors that serve in transmination and related processes.
Which ones add carbons by making C–C bonds? (note that CoASH and lipoamide carry carbons, but don't make C–C bonds)

cofactor	carbons carried & how bonds are made
SAM	one, via S_N2
Biotin	one, via a Claisen/aldol hybrid process
TPP	two (usually), via a species that is like an enamine

A good place to see cofactors at work is to study what happens to pyruvate...
- It can be decarboxylated using TPP to give acetaldehyde and then reduced to ethanol with NADH
- It can be decarboxylated and converted to Acetyl CoA via a TPP/lipoamide catalyzed process
- It can be converted to oxaloacetate via carboxylation using biotin