1. TK1656, a thermostable l-asparaginase from Thermococcus kodakaraensis, exhibiting highest ever reported enzyme activity
Shahid Mahmood Chohan, Naeem Rashid J Biosci Bioeng. 2013 Oct;116(4):438-43. doi: 10.1016/j.jbiosc.2013.04.005. Epub 2013 May 4.
Two L-asparaginase homologs, TK1656 and TK2246, have been found in the genome of Thermococcus kodakaraensis. The gene encoding TK1656 consists of 984 nucleotides corresponding to a polypeptide of 328 amino acids. To examine the properties of TK1656, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. TK1656 exhibited high asparaginase activity (2350 U mg⁻¹) but no glutaminase activity. The enzyme also displayed the D-asparaginase activity but 50% to that of L-asparaginase. The highest activity was observed at 85°C and pH 9.5. TK1656 catalyzed the conversion of L-asparagine to L-aspartatic acid and ammonia following Michaelise-Menten kinetics with a K(m) and V(max) values of 5.5 mM and 3300 mmol min⁻¹ mg⁻¹, respectively. The activation energy from the linear Arrhenius plot was found to be 58 kJ mol⁻¹. Unfolding studies suggested that urea could not induce complete unfolding and inactivation of TK1656 even at a concentration 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity.
2. Reaction of a peptide with polyvinylpyrrolidone in the solid state
Ajit Joseph M D'Souza, Richard L Schowen, Ronald T Borchardt, Jonathon S Salsbury, Eric J Munson, Elizabeth M Topp J Pharm Sci. 2003 Mar;92(3):585-93. doi: 10.1002/jps.10316.
During stability studies at high temperature (70 degrees C) and low relative humidity ( approximately 0%), the recovery of an asparagine containing hexapeptide (VYPNGA) and its known deamidation products from solid polyvinylpyrrolidone (PVP) matrices was incomplete. To determine the causes of this mass loss, formulations were prepared by lyophilizing solutions containing PVP, glycerol, and the Asn-hexapeptide in pH 7.5 phosphate buffer, followed by storage at 70 degrees C and 0% relative humidity. Asn-hexapeptide loss was mono-exponential and reached a plateau at about 30% remaining. Total recovery of the peptide and its known deamidation products was approximately 30% of peptide load. Size exclusion chromatography with fluorescence detection indicated the formation of a PVP-peptide adduct that was stable in the presence of 6 M guanidine hydrochloride. Similar stability studies using N-acetyl phenylalanine, phenylalanine ethyl ester, and N-acetyl tyrosine ethyl ester demonstrated that the reaction involves the peptide N-terminus. The adduct was disrupted in the presence of carboxypeptidase-A, suggesting the formation of an amide bond between the peptide and PVP. (15)N solid-state nuclear magnetic resonance spectroscopy using (15)N-labeled valine as a model of the peptide N-terminus showed different populations of (15)N, suggesting that noncovalent peptide-polymer interactions precede amide bond formation.
3. Hypersensitivity of rat glioma sublines with acquired ACNU resistance to L-asparaginase
Y Saito, Y Nakada, T Hotta, T Mikami, K Kurisu, K Kiya, K Kawamoto, T Uozumi J Neurosurg. 1991 Dec;75(6):930-4. doi: 10.3171/jns.1991.75.6.0930.
Cell lines resistant to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) show a high degree of collateral sensitivity to L-asparaginase. The mechanism for this phenomenon was investigated by comparing the nutritional requirements and asparagine synthetase activity of the resistant sublines to those of parent cells. Nine ACNU-resistant sublines were isolated from rat glioma 9L cells after incubation with various concentrations of ACNU in Ham's F-12 medium. The 9L cells grew independently of asparagine, developing well in asparagine-deficient Dulbecco's modified Eagle's medium. In contrast, the growth rates of all nine ACNU-resistant sublines decreased under the same conditions and required the addition of 10(-4) M asparagine for maximum growth. Asparagine synthetase activity in the ACNU-resistant cells was much lower than in the 9L cells, suggesting that the requirement for asparagine in the resistant sublines was due to reduced activity of this enzyme. A growth-inhibition assay showed that the ACNU-resistant sublines were more sensitive to L-asparaginase than 9L cells by up to 2 x 10(5)-fold. These results suggest that L-asparaginase therapy has the potential to become a new approach for treating acquired ACNU resistance.
