Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III

Chen, Xian; Tu, Chingkuang; LoGrasso, Philip V.; Laipis, Philip J.; Silverman, David N.

doi:10.1021/bi00082a004

Cited by 16 publications

(11 citation statements)

References 38 publications

(38 reference statements)

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“…The positioning and apparent functioning of Thr 325 in TrzN has a known precedent in carbonic anhydrase (CA), a protein that is in a different structural family but was observed here to have a similar active site (32)(33)(34)(35)(36). The overall folds differ markedly.…”

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confidence: 73%

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Seffernick

Reynolds

Fedorov

et al. 2010

Journal of Biological Chemistry

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Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (␤␣) 8 barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinccoordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k cat /K m , largely due to k cat , whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu 241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.There is substantial evidence that microbes rapidly evolve new enzymes to metabolize anthropogenic chemicals (1, 2). The s-triazine herbicides, such as atrazine, were first introduced into the environment 50 years ago and Ͼ2 billion pounds have been applied globally. s-Triazine compounds were initially found to be poorly biodegradable, but more rapid biodegradation is observed today (3). Many atrazine-degrading bacteria have now been isolated (4 -6). They invariably contain highly conserved genes encoding enzymes that hydrolytically displace substituents from the s-triazine ring carbon atoms to generate cyanuric acid (Fig. 1). The genes, trzN, atzA, atzB, and atzC, are found on plasmids and now are distributed globally (7, 8). These observations are consistent with the idea that a new metabolic pathway for atrazine catabolism may have evolved and spread in recent evolutionary times (9).The s-triazine herbicides, such as atrazine (2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine) and ametryn (2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine), are metabolized readily by dedicated enzymes. The enzymes have been purified to homogeneity and shown to be inactive with structurally analogous pyrimidines and other closely related compounds (10 -13). Moreover, the enzymes are isolated from bacteria...

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confidence: 73%

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Seffernick

Reynolds

Fedorov

et al. 2010

Journal of Biological Chemistry

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“…In the free enzyme, the side chain of the catalytically important residue Thr199, invariant in all mammalian carbonic anhydrases, is a hydrogen bond acceptor for the zinc-bound hydroxide involved in the hydration reaction, and a hydrogen bond donor to the carboxylate of Glu106 44. Replacing Thr199 with Ala causes a 100-fold decrease in the activity of HCA II44 and a 4-fold decrease in the activity of HCA III 45. Furthermore, Chen et al have suggested that the interaction between Thr199 and the residue at position 198 (Leu in HCA II and Phe in HCA III) is partly responsible for the large differences in activity between HCA II and HCA III 45.…”

Section: Introductionmentioning

confidence: 99%

Exploring the Molecular Origins of Protein Dynamics in the Active Site of Human Carbonic Anhydrase II

et al. 2009

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We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 → Ala (T199A) and Leu198 → Phe (L198F). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in T199A results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in L198F causes much faster dynamics. We conclude that the fast, subpicosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.

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“…To some extent this can be attributed to the increase in the pK a of the zinc-bound water, which then renders the zinc-bound hydroxide more nucleophilic in the CO 2 hydration step (Equation 1). Chen et al (31) and LoGrasso et al (32) have discussed the effect on catalysis of the mutation at residue 198. Two of these mutants, Y7H/F198L and Y7H/K64H/F198L, were somewhat unstable at pH Ͻ6 and showed evidence of decay during the course of the 18 O exchange experiments.…”

Section: Resultsmentioning

confidence: 99%

Kinetic Analysis of Multiple Proton Shuttles in the Active Site of Human Carbonic Anhydrase

Tu¹,

Qian²,

An³

et al. 2002

Journal of Biological Chemistry

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We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His 7 in HCA III. Using an 18 O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His 7 and His 64 within the active site cavity, shows the conformation of the side chain of His 64 moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His 7 . This suggests a cause of decreased proton transfer in catalysis.The carbonic anhydrases in the ␣ class include the mammalian isozymes and are all zinc-containing monomeric enzymes, generally with molecular masses near 30 kDa. These isozymes of carbonic anhydrase catalyze the dehydration/hydration of HCO 3 Ϫ /CO 2 by a two-stage or ping-pong mechanism in which the first step (Equation 1) is the reaction of bicarbonate with the enzyme containing zinc-bound water resulting in the formation of CO 2 and leaving zinc-bound hydroxide at the active site (1, 2).The second stage is the regeneration of the zinc-bound water through proton transfer (1, 2). BH ϩ represents a proton donor that is a residue of the enzyme itself or buffer in solution. In the carbonic anhydrases, the maximal velocities of catalysis and the rates of exchange of 18 O between CO 2 and water are limited by the intramolecular proton transfers indicated in Equation 2. This was initially determined by observation of a solvent deuterium isotope effect of 3.8 on k cat for hydration catalyzed by human carbonic anhydrase (HCA) 1 II, among the most efficient isozymes in the ␣ class of carbonic anhydrases (3). The solvent hydrogen isotope effect was 2.4 for 18 O exchange catalyzed by HCA III, the least efficient of the ␣ class (4). Subsequent results including pH profiles, buffer activation, and computer simulations for these isozymes support the rate-limiting nature of proton transfer in these isozymes (4 -7).For HCA II, the catalytic turnover is near 10 6 s Ϫ1 . For this isozyme the predominant shuttle residue has been identified as His 64 (3, 5), the side chain of which extends into the active site cavity without apparent interactions with other residues of the protein (8, 9). The imidazole ring of His 64 is about 7.5 Å from the zinc when this side chain is in the "in" conformation pointing toward the metal (8). This is too far for direct proton transfe...

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Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III

Cited by 16 publications

References 38 publications

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Exploring the Molecular Origins of Protein Dynamics in the Active Site of Human Carbonic Anhydrase II

Kinetic Analysis of Multiple Proton Shuttles in the Active Site of Human Carbonic Anhydrase

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