The protective outer cell wall in bacteria has D-alanine as one of the building blocks. However, met — Organic Chemistry Chemistry Question
Coenzyme chemistry
The protective outer cell wall in bacteria has D-alanine as one of the building blocks. However, metabolically only L-amino acids are available. Bacteria make D-alanine by inverting the L-alanine. The structure of L-alanine is given below:
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The abstraction of α-proton from L-alanine and reprotonation of the resultant carbanion from the opposite side appears to be a simple process. However, it is not easy to deprotonate alanine unless its NH2 group is masked and C-H is activated as an acid. Both these steps are brought about by the coenzyme pyridoxal phosphate (PLP) in the presence of the enzyme alanine racemase. The following observations made in certain model reactions will help you appreciate the role of PLP as the coenzyme.
Under favorable experimental conditions, benzaldehyde can be used as a reagent to racemize alanine. In other words, it can mask the amine group and activate the C-H of alanine making it more acidic.
Propose a stepwise mechanism for this base catalyzed racemisation of L-alanine involving benzaldehyde as the reagent.
Model Answer
Step 1: Schiff base formation
Step 2: Proton abstraction
Step 3: Reprotonation
Step 4: Hydrolysis
Compared to benzaldehyde, PLP is a somewhat complex molecule. With the help of a few carefully designed aromatic aldehydes, good insight about the role of PLP as a coenzyme can be obtained.
A few relevant structures are presented below. Underneath each, there is an indication about its activity.
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Based on this information, what inferences can you draw about the structural requirements for PLP to act as a coenzyme?
Model Answer
From the information stated in the problem, the following conclusions can be drawn:
Structure 2: Removal of the phosphate group does not hamper the activity. This indicates that the phosphate is not critical for the activity of PLP.
Similarly,
Structure 3: CH2-OH is not critical.
Structure 4: Phenolic OH is needed in the free form.
Structure 5: NO2, a well-known electron withdrawing group, causes benzaldehyde to become activated. Hence positively charged nitrogen in structure 3 must be also important for its electron withdrawing effect.
Structure 6: Electron withdrawing effect of NO2 is only effective from the para position. Introduction of this group at meta position leads to an inactive analog.
A trivalent metal ion is actually critically needed for any of the above shown compounds to display PLP-like activity without the involvement of the enzyme. Suggest a plausible explanation for the role of the metal ion.
Model Answer
Role of metal ion: The metal ion is involved in a chelation, as shown below, and provides an explanation for the critical role of the phenolic OH. The planar structure formed due to chelation assists in the electron flow.
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PLP is quite a versatile coenzyme. It participates in a variety of biologically important reactions. The activity of PLP is due to its functioning as an electron sink that stabilizes carbanions.
An important illustration of catalytic versatility of PLP is in the biosynthesis of the neurotransmitter gamma amino butyric acid (GABA). As shown below, GABA is made in a single step from L-glutamic acid. Suggest a mechanism explaining the role of PLP as the coenzyme in this particular reaction.
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Model Answer
Step 1: Schiff base formation and decarboxylation
Step 2: Tautomerization
Step 3: Hydrolysis
In yet another PLP mediated reaction, L-serine serves as a one-carbon donor in a complex process of nucleotide biosynthesis. The enzyme serine hydroxymethyltransferase degrades L-serine with the help of PLP into the simpler amino acid glycine. An important metabolic intermediate (X) is obtained as the side product in this reaction.
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Identify the one carbon metabolic intermediate formed by analyzing its PLP based mechanism.
Model Answer
Step 1: Schiff base formation followed by carbon-carbon bond scission.
Step 2: Tautomerization followed by hydrolysis
The intermediate (X) is formaldehyde (HCHO).