The catalytic activity of a series of Au monolayer protected colloids (Au MPCs) containing different ratios of the catalytic unit triazacyclononane⋅ZnII (TACN⋅ZnII) and an inert triethyleneglycol (TEG) unit was measured. The catalytic self-assembled monolayers (SAMs) are highly efficient in the transphosphorylation of 2-hydroxy propyl 4-nitrophenyl phosphate (HPNPP), an RNA model substrate, exhibiting maximum values for the Michaelis–Menten parameters kcat and KM of 6.7×10−3 s−1 and 3.1×10−4 M, respectively, normalized per catalytic unit. Despite the structural simplicity of the catalytic units, this renders these nanoparticles among the most active catalysts known for this substrate. Both kcat and KM parameters were determined as a function of the mole fraction of catalytic unit (x1) in the SAM. Within this nanoparticle (NP) series, kcat increases up till x1≈0.4, after which it remains constant and KM decreases exponentially over the range studied. A theoretical analysis demonstrated that these trends are an intrinsic property of catalytic SAMs, in which catalysis originates from the cooperative effect between two neighboring catalytic units. The multivalency of the system causes an increase of the number of potential dimeric catalytic sites composed of two catalytic units as a function of the x1, which causes an apparent increase in binding affinity (decrease in KM). Simultaneously, the kcat value is determined by the number of substrate molecules bound at saturation. For values of x1>0.4, isolated catalytic units are no longer present and all catalytic units are involved in catalysis at saturation. Importantly, the observed trends are indicative of a random distribution of the thiols in the SAM. As indicated by the theoretical analysis, and confirmed by a control experiment, in case of clustering both kcat and KM values remain constant over the entire range of x1.