Phosphorylation of adenosine or guanosine diphosphates to form ATP or GTP is one of the most important biochemical reactions. One of the enzymes that makes ATP in vivo is phosphoglycerate kinase (PGK), which transfers a phosphoryl group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, generating ATP and 3-phosphoglycerate (3-PG). PGK is a promiscuous enzyme and will also phosphorylate GDP. Both reactions require the binding of magnesium ions to the nucleotides. The binding of substrates and magnesium ions results in substantial conformational changes in the enzyme and the substrates, but these processes are not fully understood. Also, the dynamic behaviour of nucleotide-Mg complexes and the mode of coordination in aqueous phase remain elusive. Therefore, this thesis aimed to use Raman spectroscopy and Density Functional Theory (DFT) to characterise the common adenosine and guanosine nucleotides and identify the frequencies of the vibrational modes associated strongly with their phosphate groups. The study investigated the influence of magnesium binding on the spectral profile of adenosine and guanosine mono-, di-and tri- phosphates in aqueous solution at different pH values. These studies were supported by 31P NMR measurements under identical conditions. These results identified marker bands for adenosine and guanosine mono-, di-and tri- phosphates. In the absence of the enzyme, magnesium ions was found to coordinate both Î±- and Î²-phosphate groups of 5â-adenosine and guanosine diphosphates, and the Î²- and Î³-phosphate groups of 5â-adenosine and guanosine triphosphates at pH 7.0. The results also indicated that acidification does not substantially alter the coordination of magnesium ions or the phosphate binding sites but does measurably affect the vibrational modes associated with the phosphate groups in adenosine and guanosine mono-, di-and tri- phosphates. The study further applies Raman spectroscopy to explore the spectral profile of the human PGK (hPGK) in its open conformation and when closed in the presence of transition state analogues. The influence of conformational changes, isotopic substitution with 15N, and substrate binding on the Amide I and Amide III regions of the enzyme were investigated. Changes in the PGK spectral profile upon binding to ADP and 3-PG were observed and attributed to the contribution of some Î±-helices of hPGK. In the closed conformation of hPGK, both Î²-sheets and Î±-helices of Amide I and III regions are involved. The study also suggested that Raman vibrations identified at 1030-1070 cm-1 in hPGK-ADP complex could arise from ADP vibrations. The results also highlighted the role of hydrogen bonding in stabilizing the enzyme during its catalytic cycle.