Microbial and enzyme functioning depends on the quality of substrates, which strongly differ in bare soil and in the hotspots of microbial activity such as the rhizosphere and detritusphere. We established a field experiment to determine the effects of contrasting substrate quality, namely, soil organic matter, maize shoot litter (detritusphere) and maize rhizodeposits (rhizosphere) on microorganisms and their extracellular enzymes in an arable soil. Kinetic parameters (Vmax and Km) of four hydrolytic extracellular enzymes: β-cellobiohydrolase, β-glucosidase, acid phosphate and β-xylosidase were analyzed in 0–10 and 10–20 cm to elucidate the effects of substrate content on substrate affinity and catalytic efficiency (Vmax/Km). Living roots increased microbial biomass by 179% and microbial respiration by 100% compared to fallow soil. Lower enzyme affinities to substrates (e.g. 93% for β-glucosidase) in rooted soil pointed to the domination of r-strategists, which are favored in the decomposition of labile organics common in the rhizosphere. No differences in catalytic properties of cellulolytic enzymes were detected between bulk and litter-treated soil, indicating the recalcitrance of organics in both treatments. The rhizosphere and detritusphere effects on enzyme kinetics were negligible in 10–20 cm, except β-glucosidase. The reduction of Km of all enzymes in 10–20 cm versus the upper 10 cm indicated increasing substrate affinity with depth. Nonetheless, the catalytic efficiency increased from 0 to 10 to 10–20 cm (e.g. up to 420% for acid phosphatase), reflecting changes in properties and functioning of enzymatic systems. This pointed to a shift towards a more K-selected microbial community with higher affinity and more efficient substrate utilization. It also indicated the microbial adaptation to decreasing substrate contents with depth by altered enzyme functioning. Overall, the catalytic properties of cellulolytic enzymes were much more strongly affected by plants (substrate quality in the rhizosphere and detritusphere compared to bare fallow) than by depth (substrate content). Display omitted •Living roots increased microbial biomass by 179% and microbial respiration by 100% in rooted topsoil compared to fallow soil.•The catalytic efficiency (Vmax/Km) of acid phosphatase was higher in rhizosphere and detritusphere compared to fallow.•The Km reduction of all enzymes in 10–20 cm versus the upper 10 cm indicated increasing substrate affinity with depth.•The catalytic efficiency increased from 0–10 to 10–20 cm for β-glucosidase, acid phosphatase and β-xylosidase.