It was recently proposed that hydrophobic interactions control the active conformation of serine proteases in the trypsin family (Hedstrom et al. (1996) Biochemistry 35, 4515-23) rather than a charge interaction with Asp next to the active site Ser, as formerly believed. In the present study, certain sitedirected mutants of the serine protease zymogen pro-urokinase (pro-UK) and its two-chain enzymatic derivative urokinase (UK) were characterized. The results provide information on the structure-function of the catalytic domain of pro-UK/UK, which is relevant to this controversy. Mutations at Asp355(c194), which eliminated its charge, induced a 6250-fold reduction in the catalytic activity of UK. By contrast, reducing the hydrophobicity at the neoterminal Ile159(c16) of UK had relatively little effect. However, when both the hydrophobicity and the size of the side chain were reduced by a glycine substitution at this position, a major reduction (9090-fold) in the catalytic efficiency of UK occurred. This effect was related to the smaller side chain increasing the cavity and the flexibility of the N-terminus and thereby interfering with its charge interaction with Asp355(c194). A similar mechanism, rather than a change in hydrophobicity, is believed also to explain the reduction in the stabilization energy of the activation domain observed in a trypsin mutant by Hedstrom et al. (1996). Although hydrophobic interaction facilitated the charge interaction with Asp355(c194), the latter was the primary force which stabilized the active conformation of UK. The charge interaction with Asp355(c194) was also found to be the principal determinant of the intrinsic catalytic activity of single-chain pro-UK. Additionally, the findings confirmed that the KM of pro-UK for its natural substrate was significantly lower than that of UK. Since this same phenomenon was also seen with each of the mutants, the substrate binding pocket of these single-chain zymogens was better formed than that of their two-chain, enzymatic derivatives.