| dc.description.abstract | Diaminopropionate ammonia lyase (DAPAL) which belongs to the class of PLP enzymes is reported only from prokaryotes. It is involved in the removal of two amino groups from its substrate, diaminopropionate, to form ammonia and pyruvate. DAPAL from Escherichia coli (eDAPAL) and Salmonella typhimurium (sDAPAL) was cloned, over expressed and purified using either affinity chromatography or conventional procedures. It was observed that eDAPAL (90 units / mg) was comparatively less active than sDAPAL (200 units / mg). Also the enzymes with the N-terminal His tag were found to be many fold less active than the enzymes without tag. DAPAL had a characteristic absorption maximum at 414nm due to the Schiff`s linkage between PLP and the € - amino group of the active site lysine residue. The apoenzyme was prepared by reaction with L-cysteine, and the resulting thiazolidine complex was easily dialyzed. On reconstitution with PLP, complete regain of absorption spectrum and 60% activity was seen. All the three enzymes (apo-, holo and reconstituted), when subjected to gel filtration chromatography were found to be homodimers of 88 kDa. The active site lysine 78 was mutated to glutamine, and the enzyme was purified to homogeneity. In the mutant enzyme PLP continued to be bound at the active site, but in a different orientation with an absorbance maximum at 406nm. The K78Q enzyme had negligible activity as compared to the wild type enzyme confirming the role of K78 in catalysis.
Only a few of the enzymes of the class have been investigated for their unfolding pathways. Urea induced unfolding studies on sDAPAL revealed that at lower concentrations of urea there was a loss in activity due to the disruption of Schiff's linkage. No gross conformational changes were observed at these concentrations of urea as seen from fluorescence and gel filtration experiments. Increase in concentration of urea led to unfolding of the protein thereby causing a shift in fluorescence maximum from 340nm to 357 nm due to the exposure of the buried tryptophans to the less hydrophobic environment. A considerable amount of aggregation was seen at intermediate urea concentrations, which was possibly the reason for the inability of the protein to refold completely. Based on the results, a concerted mechanism for dissociation and unfolding was proposed for sDAPAL.
Aminooxy compounds, which are mechanism-based inhibitors for PLP enzymes have been used as drugs against various disorders for the last few decades. In order to probe the mechanism and efficiency with which these compounds inhibit sDAPAL, cycloserine (D and L), methoxyamine (MA) and aminooxyacetic acid (AAA) were chosen for the inhibition studies. The inhibition rates were measured by monitoring decrease in absorbance at 414nm, increase in the range of 320-330nm due to the product formation and loss of activity upon incubation with the inhibitor. It was seen that both the enantiomers of cycloserine were equally effective in disrupting the Schiff’s linkage with the second order rate constants of 15.8 and 36 M -1 sec –1 respectively. Spectral measurements showed two isosbestic points in the case of DCS and one in the case of LCS. Product of this inhibition reaction was identified to be a heat and acid stable compound namely a hydroxyisooxazole derivative of PMP. It was similar in nature to that reported from GABA aminotransferase. These results showed that unlike in the case of alanine racemase, sDAPAL could be inhibited equally well by both the enantiomers. The inhibition studies with the other two inhibitors namely AAA and MA, showed AAA to be more efficient at disrupting the Schiff’s linkage and causing inactivation of the enzyme. The visible absorbance spectrum showed a single isosbestic point in both the cases, indicative of a single step involved in the formation of the final product. The elution profile of the product of the enzymatic as well as non-enzymatic reactions on a C-18 HPLC column was similar and the product was identified to be an oxime. These inhibitors reacted with sDAPAL many fold better than the other PLP dependent enzymes and therefore these compounds can serve as potential drugs for sDAPAL. | en |
