Unraveling the Catalytic Mechanism of Nitrile Hydratases

Abstract

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants kcat and Km for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2–11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pKES1 and pKES2 values of 5.9 ± 0.1 and 9.2 ± 0.1 (kcat′ = 130 ± 1 s-1), respectively, and pKE1 and pKE2 values of 5.8 ± 0.1 and 9.1 ± 0.1 (kcat′/Km′ = (6.5 ± 0.1) × 103 s-1 mm-1), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 °C, an Arrhenius plot was constructed by plotting ln(kcat) versus 1/T, providing Ea = 23.0 ± 1.2 kJ/mol. The thermal stability of PtNHase also allowed ΔH0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH0ion data, pKES1 is assigned to βTyr68, whereas pKES2 is assigned to βArg52, βArg157, or αSer112 (NHases are α2β2-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed. To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants kcat and Km for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2–11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pKES1 and pKES2 values of 5.9 ± 0.1 and 9.2 ± 0.1 (kcat′ = 130 ± 1 s-1), respectively, and pKE1 and pKE2 values of 5.8 ± 0.1 and 9.1 ± 0.1 (kcat′/Km′ = (6.5 ± 0.1) × 103 s-1 mm-1), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 °C, an Arrhenius plot was constructed by plotting ln(kcat) versus 1/T, providing Ea = 23.0 ± 1.2 kJ/mol. The thermal stability of PtNHase also allowed ΔH0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH0ion data, pKES1 is assigned to βTyr68, whereas pKES2 is assigned to βArg52, βArg157, or αSer112 (NHases are α2β2-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed. Nitrile hydratase (NHase 2The abbreviations used are: NHase, nitrile hydratase; PtNHase, P. thermophila JCM 3095 cobalt-type nitrile hydratase; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid. ; EC 4.2.1.84), one of the enzymes in the nitrile degradation pathway, catalyzes the hydrolysis of nitriles to their corresponding higher value amides in a chemo-, regio-, and/or enatioselective manner at ambient pressures and temperatures at physiological pH (Scheme 1) (1Petrillo K.L. Wu S. Hann E.C. Cooling F.B. Ben-Bassat A. Gavagan J.E. Dicosimo R. Payne M.S. Appl. Microbiol. Biotechnol. 2005; 67: 664-670Crossref PubMed Scopus (41) Google Scholar, 2Padmakumar R. Oriel P. Appl. Biochem. 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Li Z.Y. Chem. Commun. (Camb.). 2003; 3: 386-387Crossref Scopus (25) Google Scholar, 13Dadd M.R. Claridge T.D. Walton R. Pettman A.J. Knowles C.J. Enzyme Microb. Technol. 2001; 29: 20-27Crossref PubMed Scopus (18) Google Scholar, 14Martnkov L. Klempier N. Prepechalov I. Prikrylov V. Ovesn M. Griengl H. Kren V. Biotechnol. Lett. 1998; 20: 909-912Crossref Scopus (15) Google Scholar). Therefore, the potential use of nitrile-hydrolyzing enzymes for the production of several fine chemicals is increasingly recognized. NHases are metalloenzymes that contain either a non-heme Fe(III) ion (iron-type) or a non-corrin Co(III) ion (cobalt-type) in their active site and are typically α2β2-heterotetramers (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, 6Banerjee A. Sharma R. Banerjee U.C. Appl. Microbiol. Biotechnol. 2002; 60: 33-44Crossref PubMed Scopus (370) Google Scholar, 15Endo I. Nojiri M. Tsujimura M. Nakasako M. Nagashima S. Yohda M. Odaka M. J. Inorg. Biochem. 2001; 83: 247-253Crossref PubMed Scopus (121) Google Scholar, 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar). In all known NHases, each α-subunit has a highly homologous amino acid sequence (CXYCSCX) that forms the metal-binding site. Cobalt-type NHases contain threonine and tyrosine in the -C(T/S)YCSC(Y/T)- sequence of the active center, whereas iron-type NHases contain serine and threonine (6Banerjee A. Sharma R. Banerjee U.C. Appl. Microbiol. Biotechnol. 2002; 60: 33-44Crossref PubMed Scopus (370) Google Scholar). Recently, both iron- and cobalt-type NHases have been crystallographically characterized (17Hourai S. Miki M. Takashima Y. Mitsuda S. Yanagi K. Biochem. Biophys. Res. Commun. 2003; 312: 340-345Crossref PubMed Scopus (56) Google Scholar, 18Huang W. Jia J. Cummings J. Nelson M. Schneider G. Lindqvist Y. Structure. 1997; 5: 691-699Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 19Miyanaga A. Fushinobu S. Ito K. Wakagi T. Biochem. Biophys. Res. Commun. 2001; 288: 1169-1174Crossref PubMed Scopus (200) Google Scholar, 20Miyanaga A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar, 21Deleted in proofGoogle Scholar, 22Nakasako M. Odaka M. Yohda M. Dohmae N. Takio K. Kamiya N. Endo I. Biochemistry. 1999; 38: 9887-9898Crossref PubMed Scopus (75) Google Scholar). In all structures published to date, the trivalent metal ion is six-coordinate, with the remaining ligands made up of three cysteines and two amide nitrogens (Fig. 1). Interestingly, two of the active-site cysteine residues are post-translationally modified to cysteinesulfinic acid (–SO2H) and cysteinesulfenic acid (–SOH), yielding an unusual metal coordination geometry termed a “claw setting,” and it has been shown that, unless this Cys oxidation process occurs, NHase is inactive (23Tsujimura M. Odaka M. Nakayama H. Dohmae N. Koshino H. Asami T. Hoshino M. Takio K. Yoshida S. Maeda M. Endo I. J. Am. Chem. Soc. 2003; 125: 11532-11538Crossref PubMed Scopus (47) Google Scholar, 24Dey A. Chow M. Taniguchi K. Lugo-Mas P. Davin S. Maeda M. Kovacs J.A. Odaka M. Hodgson K.O. Hedman B. Solomon E.I. J. Am. Chem. Soc. 2006; 128: 533-541Crossref PubMed Scopus (84) Google Scholar). In iron-type NHases, nitric oxide (NO) binds in place of a metal-coordinated water/hydroxide molecule, which can be photoactivated, whereas cobalt-type NHases do not bind NO (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar). Based on theoretical calculations, the in the amide of both iron- and cobalt-type NHases has that it is as an A.J. Inorg. Chem. 2001; 40: PubMed Scopus Google Scholar). The of both iron- and cobalt-type NHases has provided the the Based on these data and several studies on active-site NHase model 5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google and 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar), reaction have been (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, M. 2004; PubMed Scopus Google Scholar). In each is produced as a reaction which to the corresponding a of and synthetic data have been reported for NHases, of the which mechanism is as as the of the the of groups in and the of the metal To the active-site residues and the of protons that are transferred in the have the pH and temperature dependence of the kinetic and of the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 Based on these data, a novel catalytic mechanism is proposed. and chemicals used in this were from and were of the The the and of PtNHase was from the in The was by a and on a previously published A. Fushinobu S. Ito K. Wakagi T. Biochem. Biophys. Res. Commun. 2001; 288: 1169-1174Crossref PubMed Scopus (200) Google Scholar, 20Miyanaga A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). the A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar), which the for the and of NHase and an NHase were at in of the at the were by the of 0.1 and The were for an by by were at The was in of and by The was with and with 1 to using and and and were by and to by using PtNHase was for catalytic activity using as the A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). In this the of was by the formation of at = were on a with a and an Enzyme were using the with as the were at ± 0.1 in pH of PtNHase at pH were using as the The of each used was and the were and The kinetic and were using from to the Km value at each pH and to the kinetic were using were from a of The used in the of all were in the The pH of each used was by the of or and for by to the of the pH P. J. Am. Chem. Soc. Scopus Google Scholar). pH of the the reaction mechanism of PtNHase, the kinetic and were as a of PtNHase the of at pH and with kcat = ± 1 s-1 and Km = ± 0.1 in the of acid. values are from those reported previously = s-1 and Km = A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2–11 and was found to display maximal activity between pH 7.2 and 7.8. of and versus pH were for PtNHase and to 1 and (Fig. Enzyme and of and Enzyme Scholar, A. of Enzyme Scholar), 1) is the theoretical maximal is the theoretical maximal catalytic is the ionization of the that the of the pH the and and are the ionization constants for an and respectively, on the or of a plot of versus pH (Fig. indicated that the Km not with Therefore, plots of versus pH and plots of versus pH provided values constants for by pH ± 0.1 ± 0.1 ± pH ± 0.1 ± 0.1 ± 0.1) × 103 in a of a plot of versus pH a bell-shaped curve that be to 1 (Fig. The of the as previously Adv. 1997; Scholar), for the and of versus pH for PtNHase are that one group is on each A to 1 was which a pKES1 value of 5.9 ± 0.1 and a pKES2 value of 9.2 ± 0.1 130 ± 1 to plots of versus of versus pH to provided a pKE1 value of 5.8 ± 0.1 and a pKE2 value of 9.1 ± 0.1 (6.5 ± 0.1) × 103 s-1 (Fig. were on PtNHase using as the at pH = P. J. Am. Chem. Soc. Scopus Google Scholar) by with kcat values for were at several of and and the were as the of versus is the at of and is the in (Fig. Proton were by the data to from the is the of is the of protons transferred in the is the of protons transferred in the and is the as in and are the of and protons in the or Proton Scholar, Rev. Biochem. PubMed Scopus Google Scholar). pH the data from and the was for a that at two protons are transferred in the at this pH (Fig. Because the for theoretical inventory at an of J. Am. Chem. Soc. Scopus Google Scholar, of Proton and Scholar), of a in the of protons in the catalytic by J. Am. Chem. Soc. Scopus Google Scholar), allowed the of the data were at = at the the and the in in The and are in The of the catalytic activity of PtNHase, in a of (Fig. The plot of the of versus at of in for the reaction of and PtNHase was to that two protons are transferred the rate-limiting step at pH 7.6. However, do not between two protons transferred and Because the Km was found to be of pH the pH the for PtNHase is of and The was using as the at pH 7.6. and theoretical were for of in a of Km and kcat for was reported previously that PtNHase is stable at 60 and pH T. T. Ito K. T. J. 1997; 83: Scopus Google Scholar). data are unusual most enzymes at temperatures °C, in a in Enzyme and of and Enzyme Scholar). thermal stability the to the of the of the of was in between and 60 for PtNHase at from to these data, both Km and kcat values were by the data to the at each temperature the kcat and Km values increased with In a = the a an Arrhenius plot was constructed by plotting ln(kcat) versus A plot was that the rate-limiting step not as the temperature is increased Enzyme and of and Enzyme Scholar). the of the the for temperatures between and was to be 23.0 ± 1.2 G. T. Biochemistry. 1997; PubMed Scopus Google Scholar). Because the of an Arrhenius plot is to = were by the = Ea and = and are the and that the is to the a plot was for versus 1/T, which ΔH0 by the by R. The were by the and = for by PtNHase at ± ΔH0 ± ± ± ± ± Ea ± 1.2 in a the thermal stability of PtNHase, the pH dependence of kcat at at several pH values between and were also at three temperatures to the of the ionizing groups exhibiting the pKES1 and pKES2 values of 5.9 and respectively. data were to providing three ionization one for each A plot of ionization constants versus temperature the is the (Fig. A and Mathew J. Inorg. Chem. 2002; 7: PubMed Scopus Google Scholar). The of ionization from these data are ± and ± respectively. The of ionization for pKES1 is in the for tyrosine or residues that the pKES1 is to active-site residues of Proton and Scholar, Enzyme and of and Enzyme Scholar). The of ionization for pKES2 is in the for serine or residues it can be assigned to one of the or αSer112 of Proton and Scholar, Enzyme and of and Enzyme Scholar). catalytic for NHases have been on theoretical synthetic and kinetic and studies (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar, M. 2004; PubMed Scopus Google Scholar, Y. J. Nagasawa T. Yamada H. J. Am. Chem. Soc. Scopus Google Scholar). In the most mechanism of NHases, the nitrile of the binds to the metal center, the active-site water/hydroxide group and the metal ion to as a acid, the nitrile M. 2004; PubMed Scopus Google Scholar). In this the water/hydroxide is by an active-site mechanism was on in and the of nitriles Y. J. Nagasawa T. Yamada H. J. Am. Chem. Soc. Scopus Google Scholar). In synthetic model studies have that nitriles can with Fe(III) and Co(III) J. W. Kovacs J.A. J. Am. Chem. Soc. 2002; PubMed Scopus (47) Google Scholar, J. S. W. Kovacs J.A. J. Am. Chem. Soc. 2001; PubMed Scopus Google Scholar). Recently, the of the iron-type NHase from Rhodococcus that NO binds in place of the metal-coordinated in the S. Nakasako M. Dohmae N. Tsujimura M. Takio K. Odaka M. Yohda M. Kamiya N. Endo I. 1998; 5: PubMed Scopus Google Scholar). In the of the cobalt-type NHase from P. thermophila by the acid that a binds to the metal the metal-coordinated A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). of these studies are with the of the nitrile with the active-site metal theoretical studies have that nitriles can be in the active site of the NHase from R. and that the nitrile can bind to the active-site metal M. 2004; PubMed Scopus Google Scholar, Inorg. Chem. 2006; PubMed Scopus Google Scholar). However, to date, detailed catalytic mechanism has been for NHase, in of a of detailed kinetic To the reaction mechanism of PtNHase, the kinetic and for the of as a of PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH data with a plot of activity versus pH reported for the cobalt-type NHase from M.S. Wu S. G. B. Nelson Biochemistry. 1997; PubMed Scopus Google Scholar). of a plot of versus pH indicated that the Km not with data that the not Therefore, plots of versus pH and plots of versus pH values. of a plot of versus pH a bell-shaped curve that a pKES1 value of 5.9 ± 0.1 and a pKES2 value of 9.2 ± 0.1 130 ± The of the as previously Adv. 1997; Scholar), of the and of versus pH for PtNHase are that one group is on each data that one group be and that a group be in the in the for to of the values is for pKES1 are the of the metal-coordinated acid in NHase of A. Chow M. Taniguchi K. Lugo-Mas P. Davin S. Maeda M. Kovacs J.A. Odaka M. Hodgson K.O. Hedman B. Solomon E.I. J. Am. Chem. Soc. 2006; 128: 533-541Crossref PubMed Scopus (84) Google Scholar) and the of the group or an active-site such as Based on the temperature dependence of the ionization the most for pKES1 is The pKES1 value be the data in both and as in on the iron-type NHase from that a is to the metal at pH H. Nelson J. Am. Chem. Soc. 1993; Scopus Google Scholar). The pKES2 value be to the of a active-site or that forms a with both the and acid ligands of the active site A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). the pKES2 value be to the of αSer112 or the on which mechanism is Based on the temperature dependence of the ionization the ionization pKES2 is assigned to either an active-site or groups were from plots of versus pH it is to values on the and M. E.C. Scholar). to the plots of versus of versus pH provided a pKE1 value of 5.8 ± 0.1 and a pKE2 value of 9.1 ± 0.1 (kcat′/Km′ = (6.5 ± 0.1) × 103 s-1 have of that a group at both and pH values. to the pKE1 value is most to βTyr68, also be to the of the metal-coordinated acid. to the pKE2 value is most to also be to or βArg157, all of which are A of the catalytic mechanism of NHases that has not been to is the chemical of the rate-limiting studies are an to an of the of the rate-limiting step as as to the of catalytic Rev. Biochem. PubMed Scopus Google Scholar). are a to the is made or the whereas at Therefore, the of PtNHase using as the at pH = P. J. Am. Chem. Soc. Scopus Google Scholar) by with The is to the of the for that step the the the the with the theoretical at in the of the in which a the a plot of the of versus be is the kcat at a of and is the kcat in oxide J. Am. Chem. Soc. Scopus Google Scholar). The of the catalytic activity of PtNHase, in a of that an is in the rate-limiting PtNHase, from and the that two protons are transferred with and of the also of two protons in the data the of a from an active-site to an active-site to a and the of a from the group of the to the the of a from the group of the to an active-site by the of a from a to the to the The = is of a with a conventional to 1 Rev. Biochem. PubMed Scopus Google Scholar). is that, at pH the of in the of 5.9 ± 0.1) in the of a to which that to the The rate-limiting step in the catalytic reaction is in the of by Because PtNHase is stable at 60 °C, PtNHase the to the of the a temperature of an Arrhenius plot for the of by PtNHase indicated that the rate-limiting step not as a of temperature Enzyme and of and Enzyme Scholar). The Ea for the is 23.0 ± for PtNHase, which is of the value reported for the iron-type NHase from that formation is for the Co(III) M. A. P. J. Catal. B. 2001; Scopus Google Scholar). The of the temperature is ± whereas that for PtNHase at is ± The is of a to the of formation and on the of the the the that of the are to formation between amino and the is with the inventory data at pH of these to the of Because the Km also with the for the formation of the at were also The value that the formation of the is and that the reaction by PtNHase is However, was found to be the is highly to an with the Based on the data and the previously reported structures and theoretical and synthetic model studies (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar, M. 2004; PubMed Scopus Google Scholar, Y. J. Nagasawa T. Yamada H. J. Am. Chem. Soc. Scopus Google Scholar), a detailed catalytic mechanism for NHases can be (Fig. mechanism is on the that nitriles bind to the trivalent metal ion active which is by and theoretical studies (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar, M. 2004; PubMed Scopus Google Scholar, Y. J. Nagasawa T. Yamada H. J. Am. Chem. Soc. Scopus Google Scholar). Therefore, that the nitrile to the active-site metal the nitrile occurs, both an active-site and an active-site are for the reaction to (5Kovacs J.A. Chem. Rev. 2004; 104: 825-848Crossref PubMed Scopus (253) Google Scholar, 16Harrop T.C. Mascharak P.K. Acc. Chem. Res. 2004; 37: 253-260Crossref PubMed Scopus (158) Google Scholar, M. 2004; PubMed Scopus Google Scholar, Y. J. Nagasawa T. Yamada H. J. Am. Chem. Soc. Scopus Google Scholar). Based on sequence of the of the crystallographically characterized and iron-type NHases and several characterized NHases, that the in PtNHase is Interestingly, the αSer112 to a catalytic with and in PtNHase (Fig. catalytic is of those in S. Curr. Chem. 2004; PubMed Scopus Google Scholar). a and the tyrosine is at pH of Biochemistry. 2004; PubMed Scopus Google Scholar, Biochemistry. 2002; PubMed Scopus Google Scholar). that a catalytic in all NHases, that one or of the residues of this catalytic as that, in PtNHase is at pH and that this process is the value of an catalytic is with the in kcat is with A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar). The that Km also by of with that also to bind and the Because the in kcat is with is not that for a the which is also of the catalytic as the by of the by a nitrile the and place the in the with to the catalytic and the for the of an the Based on two protons are transferred in the which is to a transferred to the nitrile and the to with occurs, the can to an amide with a from which to protons from the amide can be by a molecule, providing the of the NHases from P. the active-site A. Fushinobu S. Ito K. Shoun H. Wakagi T. Eur. J. Biochem. 2004; 271: 429-438Crossref PubMed Scopus (73) Google Scholar, 21Deleted in proofGoogle for