Importance of the Histidine Ligand to Coenzyme B12 in the Reaction Catalyzed by Methylmalonyl-CoA Mutase

Abstract

Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the ∼1012-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast,trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000- and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site. Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the ∼1012-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast,trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000- and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site. Coenzyme B12 or AdoCbl 1The abbreviations used are: AdoCbl5′-deoxyadenosylcobalamin or coenzyme B12dAdo5′-deoxyadenosineAdoCbi5′-deoxyadenosylcobinamideAdoCbi-GDP5′-deoxyadenosylcobinamide guanosine diphosphateN-MeImN-methylimidazoledmbdimethylbenzimidazole -dependent enzymes catalyze a wide variety of isomerization reactions in which a migrating group and a hydrogen atom on vicinal carbons exchange positions. A common function of the cofactor in these reactions is to serve as a dormant source of radicals that is activated by homolysis of the organometallic Co–C bond upon substrate binding. The uncatalyzed rate for the cleavage of the Co–C bond in the cofactor free in solution is 3.8 × 10−9 s−1 at 37 °C (1Waddington M.D. Finke R.G. J. Am. Chem. Soc. 1993; 115: 4629-4640Crossref Scopus (81) Google Scholar). In contrast, the kcat for most AdoCbl-dependent enzymes is of the order of ∼102 s−1 leading to a predicted rate enhancement that is of the order of 1012-fold (2Hay B.P. Finke R.G. J. Am. Chem. Soc. 1987; 109: 8012-8018Crossref Scopus (251) Google Scholar). A member of this class of enzymes is methylmalonyl-CoA mutase, which catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA (reviewed in Ref. 3Banerjee R. Biochemistry. 2001; 40: 6191-6198Crossref PubMed Scopus (104) Google Scholar). It is distinguished by being the only family member that is found in both bacterial and mammalian organisms. Methylmalonyl-CoA mutase catalyzes a 0.9 × 1012-fold enhancement of the homolysis rate that corresponds to a lowering of the activation barrier by 17 kcal/mol at 37 °C (4Chowdhury S. Banerjee R. Biochemistry. 2000; 39: 7998-8006Crossref PubMed Scopus (83) Google Scholar). 5′-deoxyadenosylcobalamin or coenzyme B12 5′-deoxyadenosine 5′-deoxyadenosylcobinamide 5′-deoxyadenosylcobinamide guanosine diphosphate N-methylimidazole dimethylbenzimidazole Unlike the porphyrins and chlorins, other members of the family of tetrapyrrolic-derived cofactors, cobalamins are characterized by the presence of a large peripheral ornamentation appended from ring D, the dimethylbenzimidazole-containing nucleotide loop. In solution and at physiological pH, AdoCbl is six-coordinate, and the lower axial ligand is the bulky and weakly basic intramolecular base, dimethylbenzimidazole (5Lenhert P.G. Hodgkin D.C. Nature. 1961; 192: 937-938Crossref PubMed Scopus (230) Google Scholar). However, in the class I or “His-on” (3Banerjee R. Biochemistry. 2001; 40: 6191-6198Crossref PubMed Scopus (104) Google Scholar) subfamily of AdoCbl-dependent enzymes, this lower ligand is replaced by a histidine residue provided by the respective proteins (6Mancia F. Keep N.H. Nakagawa A. Leadlay P.F. McSweeney S. Rasmussen B. Bösecke P. Diat O. Evans P.R. Structure. 1996; 4: 339-350Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar,7Reitzer R. Gruber K. Jogl G. Wagner U.G. Bothe H. Buckel W. Kratky C. Structure Fold Des. 1999; 7: 891-902Abstract Full Text Full Text PDF Scopus (197) Google Scholar), while the dimethylbenzimidazole moiety is held >10 Å away from the cobalt. This ligand switch was first reported in B12-dependent methyltransferases that catalyze heterolytic cleavage of the Co–C bond (8Stupperich E. Eisinger H.J. Albracht S.P.J. Eur. J. Biochem. 1990; 193: 105-109Crossref PubMed Scopus (46) Google Scholar, 9Drennan C.L. Huang S. Drummond J.T. Matthews R. Ludwig M.L. Science. 1994; 266: 1669-1674Crossref PubMed Scopus (563) Google Scholar). In contrast, the class II or “dmb-on” enzymes retain the intramolecular ligand in their bound conformation (10Yamanishi M. Yamada S. Muguruma H. Murakami Y. Tobimatsu T. Ishida A. Yamauchi J. Toraya T. Biochemistry. 1998; 37: 4799-4803Crossref PubMed Scopus (70) Google Scholar, 11Abend A. Bandarian V. Nitsche R. Stupperich E. Retey J. Reed G.H. Arch. Biochem. Biophys. 1999; 370: 138-141Crossref PubMed Scopus (64) Google Scholar, 12Lawrence C.C. Gerfen G.J. Samano V. Nitsche R. Robins M.J. Retey J. Stubbe J. J. Biol. Chem. 1999; 274: 7039-7042Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The potential role of the lower axial ligand in labilizing the upper axial Co–C bond has been the focus of enduring debate. A popular hypothesis to explain the observed rate enhancement invokes the role of conformational distortion of the corrin macrocycle (for example, see Refs. 13Grate J.H. Schrauzer G.N. J. Am. Chem. Soc. 1979; 101: 4601-4611Crossref Scopus (161) Google Scholar, 14Marzilli L.G. Toscano J. Randaccio L. Bresciani-Pahor N. Calligaris M. J. Am. Chem. Soc. 1979; 101: 6754-6756Crossref Scopus (100) Google Scholar, 15Chemaly, S. M., and Pratt, J. M. (1980) J. Chem. Soc. Dalton Trans. 2274–2281Google Scholar, 16Ng F.T.T. Rempel G.L. Halpern J. J. Am. Chem. Soc. 1982; 104: 621-623Crossref Scopus (154) Google Scholar, 17Glusker J.P. Dolphin D. B12. 1. Wiley, New York1982: 23-106Google Scholar, 18Brown K.L. Brooks H.B. Inorg. Chem. 1991; 30: 3420-3430Crossref Scopus (71) Google Scholar). According to this “mechanochemical” mechanism for labilization of the Co–C bond, an upward flexing of the corrin ring would lead to steric crowding on the β-face thereby weakening the organometallic bond. The influence of the trans electronic effects on the cobalt-alkyl bond dissociation energies have been determined with phenylethylcobalt dimethylglyoxime model compounds in which in a series of substituted pyridines were employed as the trans ligand (16Ng F.T.T. Rempel G.L. Halpern J. J. Am. Chem. Soc. 1982; 104: 621-623Crossref Scopus (154) Google Scholar). These studies revealed a linear correlation between increasing basicity of the trans ligand and increasing stabilization of the Co–C bond, consistent with the formal reduction of Co3+ to Co2+ during homolysis. Trans steric effects were evaluated in a series of benzylcobalt dimethylglyoxime model complexes in which the bulk of the tertiary phosphine ligand was varied. An inverse correlation between size and Co–C bond dissociation energy was noted (19Geno M.K. Halpern J. J. Am. Chem. Soc. 1987; 109: 1238-1240Crossref Scopus (226) Google Scholar). These results were consistent with studies on Co-dimethylglyoxime complexes in which increasing the size of the trans ligand resulted in a corresponding lengthening of the Co–C bond (20Randaccio L. Bresciani-Pahor N. Toscano P.J. Marzilli L.G. J. Am. Chem. Soc. 1980; 102: 7373-7375Crossref Scopus (60) Google Scholar, 21Summers M.F. Toscano P.J. Bresciani-Pahor N. Nardin G. Randaccio L. Marzilli L.G. J. Am. Chem. Soc. 1983; 105: 6259-6293Crossref Scopus (93) Google Scholar). However, a study on the association of a series of substituted phosphines to AdoCbi failed to obtain any evidence for binding, indicating that alkylcobaloximes are probably poor models for the biologically relevant corrinoid cofactors (22Garr C.D. Sirovatka J.M. Finke R.G. Inorg. Chem. 1996; 35: 5912-5922Crossref Scopus (45) Google Scholar). Sirovatka and Finke have compared the thermolysis of AdoCbi in the presence and absence of N-MeIm as a mimic of the His-on class of isomerases (23Sirovatka J.M. Finke R.G. J. Am. Chem. Soc. 1997; 119: 3057-3067Crossref Scopus (68) Google Scholar). The key finding from these solution studies is that N-MeIm significantly reduces the proportion of Co–C bond homolysis from ≥98% in AdoCbl to ∼50% in AdoCbi·N-MeIm, with the remaining cleavage occurring via the heterolytic pathway, and it accelerates Co–C bond homolysis and heterolysis by a factor of 8 and 350, respectively, relative to AdoCbl. In addition, the presence of N-MeIm leads to an 870-fold and 3 × 104–fold increase in the homolytic and heterolytic rates, respectively, for Co–C bond cleavage relative to AdoCbi in the solvent, ethylene glycol. To address the role of imidazole ligation in a biological context, we have examined the effect of mutating His-610, the residue provided by the Propionibacterium shermanni methylmalonyl-CoA mutase. In this enzyme, His-610 is coordinated to the cobalt and is part of a hydrogen-bonding triad involving Asp-608 and Lys-604 (Fig. 1 and Ref.6Mancia F. Keep N.H. Nakagawa A. Leadlay P.F. McSweeney S. Rasmussen B. Bösecke P. Diat O. Evans P.R. Structure. 1996; 4: 339-350Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Our study indicates that binding of AdoCbl is more severely affected than binding of AdoCbi, the cofactor lacking the dimethylbenzimidazole ribose phosphate moiety. We propose that the low activity of the H610A and H610N mutants of methylmalonyl-CoA mutase results from weak binding of the cofactor, and that a major role of the histidine may be in dimethylbenzimidazole displacement and in organization of the active site for tight cofactor binding. AdoCbl and methylmalonlyl-CoA were purchased from was purchased from other were and were used AdoCbi was as A. Toraya T. Biochemistry. 1993; PubMed Scopus Google Scholar) and characterized by at the for at the of A from the P. methylmalonyl-CoA mutase N. Keep N.H. M.L. Leadlay P.F. Biochem. J. 1990; PubMed Scopus Google Scholar, provided by P. Leadlay at the of was the which was used as a for of mutations by the with the The were employed for and The are in the respective and that were to that of the In a site was by a to a the of the to of the and were used to the presence of the in the by nucleotide The was from by with and and and was to the with the The presence of the was by nucleotide of both as as by the of a site at in the was at the at the of The enzymes were the with cofactor a for the of wild type enzyme R. Banerjee R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was used for of in the and the affinity was replaced by a exchange on a A × was with a linear from to 1 in phosphate and methylmalonyl-CoA mutase at The enzyme was and phosphate being a exchange The enzyme was with a linear from to in phosphate at a rate of Methylmalonyl-CoA mutase at was determined the with as a activity of the mutase was determined in the at 37 °C as S. R. Banerjee R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). 1 of activity catalyzes the of 1 of succinyl-CoA at 37 The of enzymes was or with to the wild type enzyme in the for the mutants were determined by increasing the of the from 3 to in the presence of of or AdoCbl to of AdoCbl or AdoCbi to methylmalonyl-CoA mutase results in a decrease in at and has been used to the dissociation for the wild type enzyme S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). However, we were to this with the His-610 mutants to a for AdoCbl or AdoCbi of the absence of at low cofactor and of protein at high of We have this at cofactor to a effect from interactions between the cofactor and the protein S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). This with to to between binding at the active site binding and suggested that the enzymes weakened binding of AdoCbi to the mutants was a in which the was at °C by a Methylmalonyl-CoA mutase in of phosphate was employed as a were between and of AdoCbi in the at °C for The AdoCbi used in these was The change in at at of AdoCbi was by the of the of free AdoCbi in phosphate The for AdoCbi was determined 1 as S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). 1 of the binding of AdoCbl to the mutants was a However, we were to at only when high of AdoCbl were to The high from the free cofactor at these the of this provided a lower for the for AdoCbl of be when low enzyme were Methylmalonyl-CoA the H610N or the H610A were to for the wild type both mutations a influence on AdoCbl binding at most a for the cofactor and The for methylmalonyl-CoA for H610N was from the for the wild type enzyme the of the H610A a In both the for AdoCbl was mutations resulted in significant in these the was the with a kcat of s−1 at 37 °C both were significantly lower than the of s−1 at 37 °C for the wild type The low of both His-610 mutants low with wild type However, the in the catalytic between the mutants was observed in This would be low of wild type enzyme were to the observed the contribution to both mutants be In addition, the for the substrate and cofactor for the mutants was from of the wild type enzyme of for wild type and His-610 mutants of methylmalonyl-CoA 37 s−1 in a of imidazole to the to a of change the activity of the mutants indicating failure to the effects of histidine In addition, enzyme activity be when AdoCbl was replaced with AdoCbi in the in the presence or absence of imidazole as observed with wild type enzyme S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). In contrast to the wild type enzyme, which an effect of the H610A the H610N an effect The weak binding of AdoCbl to the His-610 variants of methylmalonyl-CoA mutase an of the for the The of H610A reconstituted with AdoCbl was weak and corresponding to a of AdoCbl and and as in The used to this is employed with wild type methylmalonyl-CoA mutase and AdoCbl in the and lead to of the These results that the His-610 mutants of the Co–C bond leading to enzyme a that is by the wild type A has been observed with wild type enzyme reconstituted with AdoCbi, which is in the conformation in the active site S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). of AdoCbi to wild type mutase results in an increase in the as has been observed for AdoCbl S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). In contrast, the of the of cofactor a of These in at and and revealed of AdoCbi to in the presence of the These results that the absence of the histidine ligand cleavage of the Co–C bond of AdoCbi as with AdoCbl The increase in at cofactor binding was as a function of the of free cofactor and a of for H610A (Fig. and for H610N This study was to the role of trans effects in variants of methylmalonyl-CoA mutase in which the His-610, was or effects on the cofactor dissociation and on kcat effects on substrate and cofactor the H610A and H610N mutants and in respectively, as compared with the wild type enzyme, indicating a change in the that is to be dissociation for the wild type enzyme R. R. Banerjee R. Biochemistry. 1997; PubMed Scopus Google Scholar). Our to the binding for AdoCbl by electronic a lower on the for the mutants at indicating that the dissociation has been weakened by The marked in the and for AdoCbl by the His-610 mutants is However, has been reported for the corresponding mutants in mutase, and the is the is an Biochemistry. 1997; PubMed Scopus Google Scholar). by and Biochemistry. 1997; PubMed Scopus Google Scholar), a the cofactor and substrate binding and that is a that in be or than In wild type methylmalonyl-CoA mutase, the and for AdoCbl are S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar, N. L. Banerjee R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The large between the of the by the mutants is consistent with a significant change in the binding affinity for which in by the This is by of the effect in the enzymes in contrast to that of wild type enzyme that a of S. Banerjee R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This is consistent with a as cofactor binding The of the His-610 mutants reported in this study are in contrast to the effects observed when the is the cofactor analog, S. Banerjee R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The is a in the of and we have that it by methylmalonyl-CoA mutase. of results in of the observed for the cofactor free in solution indicating that His-610 when this cofactor analog This is by the of bound by of methylmalonyl-CoA mutase reconstituted with in which the for the is and with axial ligation are The for is for and the kcat is from s−1 to s−1 at The of the enzyme in the presence and absence of high of substrate significant S. Banerjee R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the kcat is only 4-fold lower with than with the of by His-610 catalytic the by site-directed mutagenesis as in the H610A most in the H610N is from the with the cofactor analog, and the of the role of His-610 is that electrostatic interactions between His-610, and Lys-604 may play an role in by a high affinity cofactor binding site (Fig. and that the absence of this hydrogen-bonding structural in the This is by the observed increase in the rate of Co–C bond cleavage of bound AdoCbl and AdoCbi and in to wild type enzyme, of the homolysis is in the absence of substrate (4Chowdhury S. Banerjee R. Biochemistry. 2000; 39: 7998-8006Crossref PubMed Scopus (83) Google Scholar). A is suggested by the of the in the binding when the nucleotide is as in AdoCbi which is significantly than when the is as in the cofactor, AdoCbl This that the histidine may be in binding of the cofactor, in the ligand displacement that must precede docking of AdoCbl to the active site of methylmalonyl-CoA mutase (Fig. The association for the intramolecular base, dimethylbenzimidazole, in AdoCbl is reported to be at °C B.P. Finke R.G. 7: Scopus Google Scholar) and the for this ligand in AdoCbl is K.L. J.M. J. Am. Chem. Soc. Scopus Google Scholar). at physiological pH, the cofactor in the and the dimethylbenzimidazole must be to the conformation in the active site. We have characterized the of AdoCbl binding to wild type mutase by and found it to be with a of S. Banerjee R. Biochemistry. 1999; PubMed Scopus Google Scholar). In contrast, when the truncated cofactor analog AdoCbi was binding of the cofactor was the that dissociation of the dimethylbenzimidazole ligand is the in the docking of the native cofactor to the mutase active site. on the results reported on the His-610 we propose a model in which the histidine a role in the conformation and thereby catalyzes cofactor binding (Fig. The observed of with AdoCbl binding would be consistent with of a histidine of the of the of mutase determined in the absence of the cofactor M. R. B. Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and of methylmalonyl-CoA mutase in the presence of the cofactor (6Mancia F. Keep N.H. Nakagawa A. Leadlay P.F. McSweeney S. Rasmussen B. Bösecke P. Diat O. Evans P.R. Structure. 1996; 4: 339-350Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), that the is and that the ligand histidine in both is on a (Fig. The is that in the presence of the leads to a that with the nucleotide this of is in the absence of B12. binding of the cofactor to the a more from the the ligand histidine M. C. R. B. J. Biol. 2001; PubMed Scopus Google Scholar) and it for It is that the of the histidine ligand in are from by the AdoCbl-dependent methylmalonyl-CoA mutase and mutase with to cofactor binding. binding of the cofactor is by this although the catalytic of the enzyme is J.T. M. C.L. Ludwig M.L. Matthews R.G. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). The ligand histidine in is a member of a triad of that to be as a that of the cofactor between and C.L. Huang S. Drummond J.T. Matthews R. Ludwig M.L. Science. 1994; 266: 1669-1674Crossref PubMed Scopus (563) Google Scholar). An role is for the and the is in these In the significance of the His-on conformation in the of isomerases that exhibit this binding is and it has been that it may an from the probably more B12-dependent family of enzymes (3Banerjee R. Biochemistry. 2001; 40: 6191-6198Crossref PubMed Scopus (104) Google Scholar). In a of AdoCbl is in bound in the conformation A. Bandarian V. Nitsche R. Stupperich E. Retey J. Reed G.H. Arch. Biochem. Biophys. 1999; 370: 138-141Crossref PubMed Scopus (64) Google Scholar, 12Lawrence C.C. Gerfen G.J. Samano V. Nitsche R. Robins M.J. Retey J. Stubbe J. J. Biol. Chem. 1999; 274: 7039-7042Abstract Full Text Full Text PDF PubMed Scopus (62) Google N. J. Tobimatsu T. Toraya T. K. Y. N. Structure Fold Des. 1999; 7: Full Text Full Text PDF Scopus Google Scholar) that of is on the presence of the histidine dimethylbenzimidazole of most B12-dependent enzymes with their respective cofactors is in is the in and in or B12 are in the cofactor have been to to B12 enzymes, of this by proteins would to be for on the class of B12 enzymes, between the ligand at the upper axial and or replacement of the ligand at the lower axial be for cofactor binding. In of the His-610 mutants of methylmalonyl-CoA mutase in this study in with studies on the of this by reconstituted enzyme that the conformation S. Banerjee R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), a role for the histidine residue that has been We propose that the histidine residue may be in catalyzing the conformational change from the to that must precede AdoCbl docking in the active site and that the influence of the histidine residue is on cofactor binding than