enzymology An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

, a proline racemase () is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

that

catalyzes Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...

the

chemical reaction A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...

:L-proline

\rightleftharpoons

D-proline Hence, this enzyme has two

substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (aquatic environment), the earthy material that exi ...

s, L- and D-

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...

, and two products, D- and L- proline. This enzyme belongs to the family of proline racemases acting on free

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s. The

systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...

of this enzyme class is proline racemase. This enzyme participates in

arginine Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidinium, guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) a ...

and

metabolism. These enzymes catalyse the interconversion of L- and D-proline in bacteria.

Species distribution

This first eukaryotic proline racemase was identified in ''

Trypanosoma cruzi ''Trypanosoma cruzi'' is a species of parasitic euglenoids. Among the protozoa, the trypanosomes characteristically bore tissue in another organism and feed on blood (primarily) and also lymph. This behaviour causes disease or the likelihood ...

'' and fully characterized . The parasite enzyme, ''Tc''PRAC, is as a co-factor-independent proline racemase and displays B-cell mitogenic properties when released by ''T. cruzi'' upon infection, contributing to parasite escape. Novel proline racemases of medical and veterinary importance were described respectively in ''

Clostridioides difficile (bacteria) ''Clostridioides'' is a genus of Gram-positive bacteria, which includes ''Clostridioides difficile (bacteria), Clostridioides difficile'', a human pathogen causing an infectious diarrhea. Taxonomy The genus ''Clostridioides'' was created to desc ...

'' () and '' Trypanosoma vivax'' (). These studies showed that a peptide motif used as a minimal pattern signature to identify putative proline racemases (motif III*) is insufficient stringent ''per se'' to discriminate proline racemases from 4-hydroxyproline epimerases (HyPRE). Also, additional, non-dissociated elements that account for the discrimination of these enzymes were identified, based for instance on polarity constraints imposed by specific residues of the catalytic pockets. Based on those elements, enzymes incorrectly described as proline racemases were biochemically proved to be hydroxyproline epimerases (i.e. HyPREs from ''

Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common Bacterial capsule, encapsulated, Gram-negative bacteria, Gram-negative, Aerobic organism, aerobic–facultative anaerobe, facultatively anaerobic, Bacillus (shape), rod-shaped bacteria, bacterium that can c ...

'' (Q9I476), ''

Burkholderia pseudomallei ''Burkholderia pseudomallei'' (also known as ''Pseudomonas pseudomallei'') is a Gram-negative, bipolar, aerobic, motile rod-shaped bacterium. It is a soil-dwelling bacterium endemic in tropical and subtropical regions worldwide, particularly in ...

'' (), ''

Brucella abortus ''Brucella abortus'' is a Gram-negative bacterium in the family Brucellaceae and is one of the causative agents of brucellosis. The rod-shaped pathogen is classified under the domain Bacteria. The prokaryotic ''B. abortus'' is non-spore-forming, ...

'' (), ''

Brucella suis ''Brucella suis'' is a bacterium that causes swine brucellosis, a zoonosis that affects pigs. The disease typically causes chronic inflammatory lesions in the reproductive organs of susceptible animals or orchitis, and may even affect joints and ...

'' () and ''

Brucella melitensis ''Brucella melitensis'' is a Gram-negative coccobacillus bacterium from the Brucellaceae family. The bacterium causes ovine brucellosis, along with '' Brucella ovis''. Humans can become infected if they have contact with an infected animal or its ...

'' ().

Structural studies

The biochemical mechanism of proline racemase was first put forward in the late sixties by Cardinale and Abeles using the '' Clostridium sticklandii'' enzyme, ''Cs''PRAC. The catalytic mechanism of proline racemase was late revisited by Buschiazzo, Goytia and collaborators that, in 2006, resolved the structure of the parasite ''Tc''PRAC co-crystallyzed with its known competitive inhibitor - pyrrole carboxylic acid (PYC). and ; Those studies showed that each active enzyme contains two catalytic pockets.

Isothermal titration calorimetry In chemical thermodynamics, isothermal titration calorimetry (ITC) is a physical technique used to determine the Conjugate variables (thermodynamics), thermodynamic parameters of interactions in Solution (chemistry), solution. ITC is the only tec ...

then showed that two molecules of PYC associate with ''Tc''PRAC in solution, and that this association is time-dependent and most probably based on mechanism of negative cooperativity. Complementary biochemical findings are consistent with the presence of two active catalytic sites per

homodimer In biochemistry, a protein dimer is a macromolecular complex or protein multimer, multimer formed by two protein monomers, or single proteins, which are usually Non-covalent interaction, non-covalently bound. Many macromolecules, such as proteins ...

, each pertaining to one enzyme subunit, challenging the previously proposed mechanism of one catalytic site per homodimer previously proposed.

Mechanism

The proline racemase active site contains two general bases, each of them a Cys, located on either side of the alpha-carbon of the substrate. In order to work properly, one Cys must be protonated (a thiol, RSH) and the other must be deprotonated (a thiolate, RS–).

Inhibition

Proline racemase is inhibited by pyrrole-2-carboxylic acid (P2C), a

transition state In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked w ...

analogue that is flat like the transition state. P2C acts as a competitive inhibitor, due to the chemical’s similarity to the transition state of the natural proline substrate. Inhibition of the active site Cys130 and Cys300 residues prevents the conversion of proline enantiomers. Pyrrole-2-carboxylic acid (P2C) reveals the presence of one catalytic center per monomer, with two Cys residues present to perform acid/base catalysis, utilizing a carbanion stabilization mechanism. The catalytic residues are 3.5 angstroms away from the molecule of P2C.

Species distribution

Structural studies

Mechanism

Inhibition

References

Further reading