Having established the activity of TtPyNP with 1a, we conducted kinetic experiments to provide further insights into this enzymatic transformation. Although TtPyNP is inhibited by pyrimidine nucleobases such as uracil (2a), we could observe Michaelis-Menten behavior of the enzyme with 1a (Figure ). Interestingly, the apparent Michalis-Menten constant KMʹ of the phosphorolysis of 1a (KMʹ = 3.37 mM) indicated that TtPyNP has a much lower affinity for 1a compared to natural nucleosides like uridine or thymidine (KM < 1 mM), suggesting that productive binding of the modified substrate 1a might present a challenge due to the increased steric bulk. In addition to a lower affinity for 1a, TtPyNP also displayed a lower rate constant compared to uridine (0.59 vs 5.05 s -1 for 1 mM substrate at 60 °C and pH 9) which showed a similar temperature-dependence as indicated by phosphorolysis experiments at different temperatures monitored by UV spectroscopy (Figure ). Collectively, these results demonstrate that, unlike other nucleoside phosphorylases, TtPyNP selectively converts the 4ʹ-methylated nucleoside 1a to the corresponding sugar phosphate 3, albeit with a lower rate constant and substrate affinity compared to the native substrates.