Enzyme - a protein that catalyzes reactions in a biological system. Enzymes catalyze reactions by providing a template upon which a substrate can reside, be distorted into a reaction shape, and coerced into bond-braking or bond-forming reactions. Enzymes are incredibly specific, meaning, their catalytic action is highly dependent upon a certain complementarity between the enzyme active site (business section) and the substrate. The interaction between a substrate and an enzyme to form its initial complement is called the enzyme-substrate complex (ES complex) or the Michaelis complex. The manner in which a substrate - enzyme interaction occurs depends greatly on the same interactions discussed earlier for normal receptor-drug interactions namely, ionic bonds, H-bonding, etc. Therefore the strength or binding of a substrate-enzyme complex may be dependent on several criteria.
 

How does an enzyme catalyze a reaction? In general, catalysts stabilize the transition state energy relative to the ground state, and a net decrease in G‡ is responsible for the rate acceleration.
 
 

An example of a transition state interaction in an enzyme (to be drawn in class):
 
 
 
 
 
 
 
 
 
 
 

Mechanisms of enzyme catalysis: the interactions and mechanisms that enzymes use to effect a reaction. Once the enzyme has "captured" the substrate, there are numerous mechanisms by which the enzyme can convert the substrate to products. These mechanisms include:
 

approximation - when an enzyme serves as a precise locking template such that the substrate is close to the reaction center (reactive groups). The locking results in a loss of rotational freedom of the substrate. Because the reactive centers are now very close to the substrate the reaction becomes first order rather than second order (as free in solution). One can think of this effect as the enhancement one would expect in an intramolecular reaction as opposed to an intermolecular.

covalent catalysis - some enzymes use a nucleophilic side chain of an amino acid residue to form covalent bonds to the substrate. A second substrate (usually water) can then react with this covalent attachment to form products. This occurs with cholinesterases.

general acid-base catalysis - reactions in enzymes that are promoted by a proton or hydroxide group. Usually general acid catalysis or general base catalysis is operative, however, both mechanisms may "gang up" on a substrate to induce the catalysis.
 
 
 

General-acid catalysis
 
 

General-base catalysis
 
 

General acid and base catalysis
 
 

electrostatic catalysis - an enzyme catalyzes a reaction by destabilization of the ground state of substrate and products and by stabilization of the transition state. If during the binding of a substrate to an enzyme active site an interaction occurs that causes a partial charge to develop (i.e., addition of a nucleophile to a carbonyl to form the C-O ) the enzyme can stabilize this pre-reaction by having a cationic group in the right position to electrostatically stabilize the negative group.
 

strain/distortion - tough to visualize but the general concept is simple. Most small ring compounds, for example epoxides, are more reactive than their straight chain (ether) brethren owing to ring strain induced by the fact that carbons prefers tetrahedral as sp³ (in epoxides the angle is 60 degrees). Therefore, if enzyme could distort a substrate into a strained form, the substrate would become more reactive.
 
 

This section is intended to answer the question, "Why are enzymes important receptor groups to consider in the construction of medicinally active agents and how can they be controlled?"
 

Many diseases occur because of a deficiency or excess of a specific metabolite or substrate in the body. If the excess or deficiency can be made right via controlling the enzyme that produces the aberrant concentration then enzymes are an ideal target for drugs. Specifically, drugs that shut down or, in part, control enzyme action are termed inhibitors or inactivators.
 

Enzyme inactivator - when he interaction between a compound and an enzyme is irreversible
 

Enzyme inhibitor - when the interaction between a compound and an enzyme slows down or blocks enzyme catalysis but is not irreversible.
 
 
 

Many drugs function as inhibitors/inactivators. But, what specifically happens when an enzyme is disabled by drug action?
 

• substrate cannot be transformed to product
• products are not produced
• secondary substrates (the product of the failed transformation) cannot move to secondary products; a cascade may be interrupted.

 

    A. Cell has a deficiency of the substrate for a target enzyme

    B. As a result of that deficiency a disease results.

    C. Therefore, inhibition of the enzyme prevents degradation of the substrate thereby increasing its net concentration. Disease cured! Nobel Prize! Trip to Bahamas!
 

A specific example of this situation
 

- onset of seizures that results from diminished gamma amino butyric acid (GABA) in the brain

- inhibition of GABA aminotransferase leads to an anticonvulsive effect (b/c GABA is not being converted into something else, its concentration builds up)
 

Consider also foreign organisms, parasites or tumor cells. Inhibition of one of the essential enzymes can prevent important metabolic processes from taking place, resulting in inhibition or growth or replication of the organism or aberrant cell. For example: the inhibition of bacterial alanine racemase prevents biosynthesis of bacterial cell walls -> the bacteria dies.
 

The use of drugs to combat foreign organisms or aberrant cells is called chemotherapy. An approach that has had excellent historical and current utility. Why do we not call antibiotics chemotherapy?
 
 

First of all, why devise an enzyme inhibitor that is reversible? Duration of effect!!
 

A. Mechanism of reversible inhibition
 

1. Competitive inhibitors: The most common drugs are those that compete with the substrate for the active site binding. Known as competitive inhibitors -> usually have structures similar to that of the native substrate or product.
 

Like a substrate, inhibitor [I] can form a complex with an enzyme. The equilibrium constant K_i (K_off/k_on) is a dissociation constant for breakdown of the EI complex. Therefore, the smaller the K_i value for I, the better the inhibitor.
 
 

When a competitive inhibitor binds at the active site, it prevents binding of substrate and product does not form. In some cases, an inhibitor may be converted into "useless products," while still preventing substrates from binding.
 

2. Non-competitive inhibitors - these inhibitors usually bind at a site other than the active site possibly causing a conformational change or structural variation that prevents substrate from binding. Typically an allosteric interaction.
 
 

In the figure above, two types of non-competitive interactions are depicted. In path A, a non-competitive inhibitor binds to the entryway to the active site blocking possible access by the substrate. There is no structural change to the enzyme other than that imposed by the binding.- certain plant, animal and bacterial toxins work in this way. In path B, a non-competitive inhibitor binds at an allosteric site causing a conformational change in the enzyme, which in this depiction, collapses the active site region making it inaccessible or inoperable toward substrate.
 
 
 

Deoxyribonucleic acid (DNA): is the polynucleotide that carries genetic information. It is therefore, an essential macromolecule and potential receptor for drug interaction. There are unfortunately few chemical/structural differences between human DNA and DNA from other cells (e.g., cancer) and specific drug targeting or specificity is difficult to achieve. Unlike drugs that act on enzymes or protein receptors that operate via specific mechanisms or essential groups, there are no principal mechanisms by which DNA-based drug can act. The principal feature that makes cancer cells different from normal cells is their ability to undergo uncontrolled cell division - this feature requires constant mitosis and the need for a steady supply of DNA and DNA precursors. Cancer chemotherapies or cytotoxic substances, therefore have targeted the shutdown of DNA knowing full well that "normal" DNA will also be shut down with the understanding that the patient will recover from having normal cells shut down for a limited amount of time. Cancer DNA drugs are quite useful against rapidly diving cell lines like leukemias and lymphoma. They are less useful against solid tumors where there is usually only a fraction of rapidly dividing cells.
 

How is the well-known toxicity of DNA-drug expressed. Usually in parts of body that also rely upon rapid cell division ---> bone marrow, GI tract, mucosa, hair, etc.
 
 
 

DNA Structure and Properties: Please review your UG biochem or organic book for precise structural features. A brief review of DNA structure is presented. DNA is a polymer of purine and pyrimidine bases (as shown) linked to a 5' phosphate group through a deoxyribose sugar. The carbon chain is numbered starting with the anomeric center (1) until it reaches the exocyclic methylene (5). Thus, the phosphate groups are connected to the 2-deoxyribose ring at the 3' and 5' positions.
 
 

The purine bases are adenine and guanine
 
 

The pyrimidine bases are cytosine and thymine
 
 

DNA features of importance to medicinal chemistry
 

• the ratio of purine (A/T) to pyrimidines (C/G) are always equal to 1:1.
• the bases are stacked on "top" of each other in the double stranded macromolecule
• the nucleotide bases are hydrogen-bonded to each other; A with T and C with G

 

 
 
 
 
 

• right handed rotation between adjacent base pairs (about 36^o) produces a double helix with 10 bases per turn.
• the glycosidic bonds that connect the base pair to its sugar ring are not directly opposite each other and therefore the two sugar-phosphate backbones of the double helix are unequal along the helical axis ----> major and minor grooves.

 

All the bases are on the inside of the double helix and the sugar-phosphates on the outside. This makes the bases close to each between strands. The need to conserve space encourages the formation of the best hydrogen bonded pairs (A-T & C-G) and leads to complementary base pairing.

For strands pointing in the 5' to 3' direction, a complementary 3' to 5' strand is required. For example,
 
 

Major groove: filled with base pair nitrogen and oxygen atoms with project inward from the sugar-phosphate backbones toward the center of the DNA.
 

Minor groove: filled with nitrogen and oxygen of base pairs that project outward from the sugar-phosphate backbones toward the outer edge of the DNA.
 

BASE TAUTOMERISM
 

Hydrogen bonding plays a critical role in the structure of DNA and therefore it is important to understand the importance of the tautomeric (hydrogen equilibrium between heteroatoms) contributions of the nucleic bases. The more stable tautomeric forms of the amino-containing nucleic bases (adenine, cytosine and guanine) is the amino form (left) and not the imino form. The major tautomeric form of thymine is the keto form (left) and not the enol form.
 
 

Although there is not nearly enough time in this course to present and detail the entire biosynthetic pathway for the purine and pyrimidine bases, a brief outline of their origins and some key transformations are useful to review. Think how or where in the pathway drugs may interact or interrupt the biosynthesis - these are good targets to reduce DNA production.
 
 

reagent legend:

a. glycine, ATPADP, b. N-formyl tetrahydrofolate, c. glutamine glutamate, d. aspartate, e. loss of fumarate, f. N¹⁰-formyl tetrahydrofolate, g. -H₂O, h. aspartate, GTPGDP, i. adenoylsuccinase; loss of fumarate, j. inosinate dehydrogenase, k. glutamineglutamate
 
 

There are three general types of compounds that interact with DNA: intercalators, alkylators and strand breakers.
 

Intercalators: a non-covalent interaction between a drug and DNA in which the drug is held perpendicular to the axis. The drug is generally aromatic or heteroaromatic (flat) and finds its way between base pairs by "stacking." To accommodate the drug, the base pairs distort and separate vertically stretching the sugar-phosphate backbone.
 
 

Complex of ethidium bromide (an intercalating reagent) with DNA (above)
 

Intercalation is energetically favored because the energy that holds the intercalated molecule must be greater than normal base stacking (otherwise the drug would be let go). Note that intercalation does not disrupt normal DNA H-bonding. However, it can destroy the regular helical structure unwinding the DNA at the binding site and importantly, interferes with DNA-binding enzymes (polymerases, topoisomerases, etc.)
 

Intercalation is not a stand alone phenomena in drug action - it is normally the first step in several stages that lead to DNA damage. The steps can be intercalation, interaction with DNA base pairs, interaction with protein, and non-productive protein-DNA interaction. Because they disrupt DNA structure which is essential to cell proliferation many intercalator drugs are aimed at cancer.
 
 
 

Alkylators: alkylation represents an irreversible (mostly) binding to DNA. In organic chemistry, we teach "substitution" reactions by concentrating on the substrate, nucleophile, leaving group and product. The nucleophile [unfortunately] is given less structural attention because we learn that it is largely there to modify the "important" substrate. In reality, biological substitution reactions regard the substrate (the alkylating agent) as minor and the nucleophile as the "important" species. The biological approach takes into account that the nucleophile is a macromolecule whose structure and function can be altered by even a minor alteration. A review of what "alkylation" means is presented first.
 
 

By definition, alkylation means the transfer of an alkyl group and the reaction must take place at physiologic pH (approx. 7.4). Two reactions are shown above: neutral substrate; charged nucleophile and neutral-neutral. There is a clear difference in the nature of the product.
 

Common functional groups that serve as alkylating agents -

alkyl iodides, bromides, and other group leaving groups attached to primary alkanes: methyl and ethyl iodide (CH₃I, CH₃CH₂I), methyl triflate (Me-OSO₂CF₃)

Michael acceptors: acrylates (CH₂=CH-CO₂R; R = H, alkyl, aryl), alkynoates (R-CC-CO₂R)

small rings: epoxides, azepines, episulfides
 

What general biological nucleophilic groups can be modified by an alkylating agent?
 

thiolates> amino > phosphate > carboxylate
 

for DNA: guanine N-7 > adenine N-3 > adenine N-1 > cytosine N-1
 
 

Thus, alkylation of guanine results in the formation of an N-7 "conjugate." The consequences of the alkylation will be discussed later in the semester in cancer therapy.
 
 

DNA strand breakers: Some DNA reactive molecules initially intercalate but under certain circumstances generate radical (C) species that react with the sugars residues of DNA causing a breakdown of the ribose group and DNA strand scission.

The anticancer drug bleomycin acts as a strand breaker and its structure and mechanism is covered as an example. In brief, bleomycin contains a "chelating domain" made up of a pyrimidine, b-amino alanine and b-hydroxyimidazole that captures an iron (II) in a complex. This complex interacts with O₂ to give a ternary complex that generates the reactive radical species. Bleomycin also contains a dithiazole domain that intercalates DNA and a sugar domain that is likely responsible for cellular recognition and uptake.