Kinetics study reveals interconnected relationships of component activities in trifunctional RNA capping enzymes

In mammalian cells, the mRNA 5' caps possess important functions such as efficient protein translation and evasion of innate immune surveillance. The primary cap structure, m⁷Gppp- or Cap-0, is generated by three enzymatic activities in tandem-RNA 5' triphosphatase (TPase), mRNA guanylyltransferase (GTase), and mRNA cap N7-guanine methyltransferase (N7-MTase). In large DNA viruses such as poxviruses and giant viruses, the three activities are consolidated into a multifunctional protein with distinct structural domains. Although widely used in mRNA synthesis in vitro, detailed mechanistic studies of these RNA capping enzymes have been limited. Here, we performed kinetic studies on Vaccinia virus (VCE) and Faustovirus (FCE) capping enzymes using a high-throughput capillary electrophoresis assay that simultaneously quantifies all substrates and products in the three reactions. We found that GTase is the rate-limiting reaction of both capping enzymes; however, in VCE, the TPase is much faster than N7-MTase, whereas the rate of the two activities is largely comparable in FCE. We further show that the GTase of VCE is slower in the forward direction than the reverse under isolated single-turnover conditions, and that the presence of N7-MTase activity increased the observed rate constant of the GTase reaction under multiple-turnover conditions. In contrast, the GTase of FCE is slightly faster in the forward direction, and the N7-MTase activity does not influence the FCE GTase reaction kinetics. Global simulation of full reactions across reaction conditions further identifies the rate-limiting steps of the individual enzyme activities and provides a framework to describe their interdependence.