Characteristic isotope fractionation patterns in S -triazine degradation have their origin in multiple protonation options in the S -triazine hydrolase trzn

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 (ε_carbon = -5.0‰ ± 0.2‰ (atrazine/TrzN), ε_carbon = -4.2‰ ± 0.5‰ (atrazine/TrzN-E241Q), ε_carbon = -2.4‰ ± 0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (ε_nitrogen = 2.5‰ ± 0.1‰ (atrazine/TrzN), ε_nitrogen = 2.1‰ ± 0.3‰ (atrazine/TrzN-E241Q), ε_nitrogen = 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 (ε_sulfur = -14.7‰ ± 1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (ε_sulfur = -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.