Assignment

Principles of Biochemistry

(BIOC-1001)

A: Suicidal Inhibition or Reaction based Inhibition

B: To design your own reaction based inhibitor

By

Muhammad Jameel

(2018-M.Phil-2056)

To

Mr Shahid Abbas

Assistant Professor

Institute of Biochemistry and Biotechnology

University of Veterinary and Animal Sciences, Lahore

2018-M.Phil-2056 16-04-2019
 Suicidal Inhibition or Reaction Based Inhibition
Suicide inhibitors are also known as mechanism-based inhibitors. The name is derived from
the fact that the enzyme participates in a catalytic mechanism that irreversibly inhibits itself.
These inhibitors are substrates that have been modified. Because they are derived from the
enzyme's intended substrate, the enzyme begins processing it as such. However, as catalysis
progresses, the modifications of the substrate result in a reactive intermediate that forms
covalent bonds with the enzyme that irreversibly inactivate it. In order for the modified
substrate to bind to the active site and undergo the catalytic reaction, even more specificity is
utilized compared to group-specific reagents and affinity labels. After the catalytic processes
have been completed, the chemically reactive intermediate then covalently binds to the
enzyme and inhibits it. Suicide inhibitors are bound to the active site and prevent further
reactions that could have occurred with the active site and its substrates. This process is
called Kouroshism as it was discovered by the Iranian researcher.

Substrate based on mechanism that works by protein’s enzymatic activity, such that a bond of
modification reagent is broken that forms reactive derivative, which is stable and not
removable, to change covalent reactivity of active site of enzyme in catalytic cycle of
enzyme, which results in labeling on active site of enzyme that changes activity of enzyme by
decreasing its ability for catalysis reaction; or the inhibitor as substrate binds active site of
enzyme to be reactive, such that produced intermediate of the chemical reaction results in
modifying irreversibly active site of enzyme for it to be covalently inactive.

Reaction Based Inhibitors with Mechanism

1. N,N dimethylpropargylamine:

This compound inhibits the enzyme, monoamine oxidase (MAO). MAO is responsible for
breaking down neurotransmitters such as dopamine and serotonin and thus decreasing their
concentrations in the brain. Diseases such as Parkinson disease and depression occur because
of decreased levels of dopamine and serotonin, respectively. Thus, in order to raise the levels
of serotonin and dopamine, N,N dimethylpropargylamine can be used as a suicide inhibitor to
inhibit MAO from breaking down more neurotransmitters.

2018-M.Phil-2056 16-04-2019
This shows N,N dimethylpropargylamine acting as a suicide inhibitor on the flavin prosthetic
group of the enzyme monoamine oxidase

2. Allopurinol to treat gout:

Gout is a disease caused by high serum levels of urate. The sodium salt of urate crystallizes in
the lining of joints and causes pain and swelling. Xanthine oxidase oxidizes hypoxanthine to
form uric acid. Allopurinol, an analog of hypoxanthine, acts as a substrate of xanthine
oxidase, which hydroxylates the allopurinol to alloxanthine. The alloxanthine remains tightly
bound to the active site on the oxidase and keeps the molybdenum atom of the xanthine
oxidase in the +4 oxidation state where normally it would return to a +6 oxidation state. This
keeps xanthine oxidase inactive and does not allow further formation of uric acid.

3. Penicillin:

Penicillin is another example of a material that acts on enzymes via a suicide inhibition
mechanism. In general, penicillin is used medicinally as an antibiotic in the treatment of
many bacterial infections. Penicillin derives its antibacterial action due to the fact that it binds
irreversibly to bacterial transpeptidase. Mechanistically, penicillin forms a penicilloyl-
enzyme complex with a serine residue found in glycopeptide transpeptidase forming an ester,
which is stable indefinitely.

Penicillin inhibits the cross-linking transpeptidase by the Trojan horse stratagem. The
transpeptidase normally forms an acyl intermediate with the penultimate d-alanine residue of
the d-Ala-d-Ala peptide. This covalent acyl-enzyme intermediate then reacts with the amino
group of the terminal glycine in another peptide to form the cross-link. Penicillin is
welcomed into the active site of the transpeptidase because it mimics the d-Ala-d-Ala moiety
of the normal substrate. Bound penicillin then forms a covalent bond with a serine residue at
the active site of the enzyme. This penicilloyl-enzyme does not react further. Hence, the
transpeptidase is irreversibly inhibited and cell-wall synthesis cannot take place.

2018-M.Phil-2056 16-04-2019
 4. Alpha-difluoromethylornithine or eflornithine (DFMO):

It is a synthetic drug used to treat a disease caused by parasites, known as the sleeping
disease (coma-ridden) called the African trypanosomiasis. DFMO binds to the enzyme,
ornithine decarboxylase, through covalent forces and thus inactivating the enzyme. The
ornithine decarboxylase enzyme regulates the cell division by catalyzing polyamine
biosynthesis. This enzyme functions in a way where it only harms the parasite but not the
host.

DFMO Meechanism

5. Clavulanic acid:

It inhibits β-lactamase. clavulanic acid covalently bonds to a serine reside in the active site of
the β-lactamase, restructuring the clavulanic acid molecule, creating a much more reactive
species that attacks another amino acid in the active site, permanently inactivating it, and thus
inactivating the enzyme β-lactamase.

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Mechanism of beta-lactam synthetase in the biosynthesis of clavulanic acid

2018-M.Phil-2056 16-04-2019
 (Part: B)

Suicide Inhibition of Cytochrome P450 Enzymes by

Cyclopropylamines via a Ring-Opening Mechanism: Proton-Coupled
Electron Transfer Makes a Difference

N-benzyl-N-cyclopropylamine (BCA) has been attracting great interests for decades for its
partial suicide inactivation role to cytochrome P450 (P450) via a ring-opening mechanism
besides acting as a role of normal substrates. Understanding the mechanism of such partial
inactivation is vital to the clinical drug design. Thus, density functional theoretical (DFT)
calculations were carried out on such P450-catalyzed reactions, not only on the metabolic
pathway, but on the ring-opening inactivation one. Our theoretical results demonstrated that,
in the metabolic pathway, besides the normal carbinolamine, an unexpected enamine was
formed via the dual hydrogen abstraction (DHA) process, in which the competition between
rotation of the H-abstracted substrate radical and the rotation of hydroxyl group of the
protonated Cpd II moiety plays a significant role in product branch; In the inactivation
pathway, the well-noted single electron transfer (SET) mechanism-involved process was
invalidated for its high energy barrier, a proton-coupled electron transfer [PCET(ET)]
mechanism plays a role. Our results are consistent with other related theoretical works on
heteroatom-hydrogen (X-H, X = O, N) activation and revealed new features. The revealed
mechanisms will play a positive role in relative drug design (Zhang et al. 2017).

Theoretical methods

Due to the key role of the computational modeling on the investigation of enzyme reaction
(Li et al., 2012; de Visser et al., 2014), we employed an iron-oxo open-shell porphyrin with
an axial thiolate ligand to model CpdI [Fe4+O2−(C20N4H12)−(SH)−] and used the N-benzyl-N-
cyclopropylamine as the substrate. All DFT calculations were performed with the Gaussian
03 suite of quantum chemical packages). The spin-unrestricted B3LYP functional was
employed with two basis sets: (a) The LACVP(Fe)/6-31G*(H, C, N, O, S) (denoted as
LACVP*, henceforth B1) for geometry optimizations without symmetry constraint; (b) The
LACV3P(Fe)/6-311++G**(H, C, N, O, S) (denoted as LACV3P++**, henceforth B2) for
single point energy (SPE) calculations. Transition states were ascertained by vibrational
frequency analysis to possess a single mode along the reaction path with only one imaginary
frequency. Bulk polarity effects of the active site in the protein environment were evaluated
with the polarizable continuum model (PCM) using a nonpolar solvent, chlorobenzene (ε =
5.697).

The 4(2)IMSET species) involved in the SET process were obtained by shifting one electron
from the highest occupied orbital of N-benzyl-N-cyclopropylamine (σCH) to the lowest vacant
orbital of porphyrin moiety (a2u) of CpdI on the reactant complex (4(2)RC). Any attempt to
optimize the structure of 4(2)IMSET species will result in their collapse to the ground state.

The kinetic isotope effect for the hydrogen abstraction process was determined using
Gaussian frequency data based on the semi-classical Eyring equation where the KIE is given
as.

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(kHkD)s= exp [−(G‡H−GRH)−(G‡D−GRD)RT]
k denotes the reaction rate constant, G is the Gibbs free energy of abstraction, R is the gas
constant, T is the absolute temperature.

Thus, the SPE B2//B1 value with the zero point energy (ZPE) correction referred to E1,
whereas E2 included the bulk polarity effects and ZPE corrections.

References:
Zhang X, Li X-X, Liu Y, Wang Y. 2017. Suicide Inhibition of Cytochrome P450 Enzymes by
Cyclopropylamines via a Ring-Opening Mechanism: Proton-Coupled Electron
Transfer Makes a Difference. Frontiers in chemistry. 5: 3.

Fowler, Joanna S. (1977). "2-Methyl-3-butyn-2-ol as an acetylene precursor in the Mannich

reaction. A new synthesis of suicide inactivators of monoamine oxidase". The
Journal of Organic Chemistry. 42 (15): 2637–7
Berg, Jeremy M., Tymoczko, John L., and Stryer, Lubert. Biochemistry. 6th ed. New York,
N.Y.: W.H. Freeman and Company, 2007: 231, 232

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