s-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by Arthrobacter aurescens TC1 (i) delivered highly characteristic (13C/12C, 15N/14N) fractionation trends for pathway identification and (ii) suggested that the s-triazine ring nitrogen was protonated in the enzyme s-triazine hydrolase (TrzN) where (iii) TrzN crystal structure and mutagenesis indicated H+-transfer from the residue E241. This study tested the general validity of these conclusions for atrazine and ametryn with purified TrzN and a TrzN-E241Q site-directed mutant. TrzN-E241Q lacked activity with ametryn; otherwise, degradation consistently showed normal carbon isotope effects (εcarbon = -5.0‰ ± 0.2‰ (atrazine/TrzN), εcarbon = -4.2‰ ± 0.5‰ (atrazine/TrzN-E241Q), εcarbon = -2.4‰ ± 0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (εnitrogen = 2.5‰ ± 0.1‰ (atrazine/TrzN), εnitrogen = 2.1‰ ± 0.3‰ (atrazine/TrzN-E241Q), εnitrogen = 3.6‰ ± 0.4‰ (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (εsulfur = -14.7‰ ± 1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (εsulfur = -0.2‰ ± 0.0‰, pH 1.75), indicating rate-determining C-S bond cleavage in TrzN. Our results highlight a robust inverse 15N/14N fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN.