The mobilization of soil nutrients bound in organic matter is largely mediated by enzymes derived from plants, soil microorganisms and animal residues. Land-use change alters important soil characteristics that may affect the activities of soil enzymes. However, mechanistic understanding of how land use and management practices influence the catalytic properties of enzymes in top- and subsoil are still scarce, especially in African ecosystems. We linked catalytic properties i.e. substrate affinity constant (Km) and maximum reaction rate (Vmax), determined by Michaelis–Menten kinetics, to a set of environmental and microbial variables in the soils of a land-use sequence (6 ecosystems) ranging from natural forests to agricultural fields at Mt. Kilimanjaro. The sensitivity of Km and Vmax of four extracellular hydrolytical enzymes, β-galactosidase, cellobiohydrolase, phosphatase and chitinase to changing environmental conditions were tested by fluorogenic substrates in topsoils and subsoils. The β-galactosidase activity increased with increasing soil depth. Other extracellular enzyme (cellobiohydrolase, phosphatase and chitinase) activities decreased with depth. The affinity of enzymes to substrates was higher in soils of natural compared to agricultural ecosystems: i.e. higher under forests than under cropland. The activity of β-galactosidase, cellobiohydrolase and chitinase enzyme were highest in lower mountain forest and grassland (less disturbed ecosystems). This indicated that changes in land use and management practice not only affects enzyme activity but also controls enzyme kinetics (Km and Ka) thus pointing towards the expression of different enzyme systems. Therefore, we concluded that anthropogenic activities result in alteration of C and nutrient cycling by affecting microbial activities and enzymes catalytic properties.