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 (C-13/C-12, N-15/N-14) 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 (epsilon(carbon) = -5.0 parts per thousand +/- 0.2 parts per thousand(atrazine/TrzN), epsilon(carbon) = -4.2 parts per thousand +/- 0.5 parts per thousand(atrazine/TrzN-E241Q), epsilon(carbon) = -2.4 parts per thousand +/- 0.3 parts per thousand (ametryn/TrzN)) and inverse nitrogen isotope effects (epsilon(nitrogen) = 2.5 parts per thousand +/- 0.1 parts per thousand (atrazine/TrzN), epsilon(nitrogen) = 2.1 parts per thousand +/- 0.3 parts per thousand (atrazine/TrzN-E241Q), epsilon(nitrogen) = 3.6 parts per thousand +/- 0.4 parts per thousand (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (epsilon(sulfur) = -14.7 parts per thousand +/- 1.0 parts per thousand, ametryn/TrzN) than in acidic ametryn hydrolysis (epsilon(sulfur) = -0.2 parts per thousand +/- 0.0 parts per thousand, pH 1.75), indicating rate-determining CS bond cleavage in TrzN. Our results highlight a robust inverse N-15/N-14 fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN.