Phylogenetic Study of the Evolution of PEP-Carboxykinase

Phosphoenolpyruvate carboxykinase (PCK) is the key enzyme to initiate the gluconeogenic pathway in vertebrates, yeast, plants and most bacteria. Nucleotide specificity divided all PCKs into two groups. All the eukaryotic mammalian and most archaeal PCKs are GTP-specific. Bacterial and fungal PCKs can be ATP-or GTP-specific but all plant PCKs are ATP-specific. Amino acid sequence alignment of PCK enzymes shows that the nucleotide binding sites are somewhat conserved within each class with few exceptions that do not have any clear ATP- or GTP-specific binding motif. Although the active site residues are mostly conserved in all PCKs, not much significant sequence homology persists between ATP- and GTP-dependent PCK enzymes. There is only one planctomycetes PCK enzyme (from Cadidatus Kuenenia stuttgartiensis) that shows sequence homology with both ATP-and GTP-dependent PCKs. Phylogenetic studies have been performed to understand the evolutionary relationship of various PCKs from different sources. Based on this study a flowchart of the evolution of PCK has been proposed

Continuous Spectrophotometric Assay for β-Glucuronidase

A continuous spectrophotometric assay has been developed for detecting β-glucuronidase activity. In the assay, Para-nitrophenyl β-d-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 × 10−4 U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies