Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys146 → Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys229 in native aldolase. Mutation of conserved active site residue Lys146 to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr363 hydroxyl within 2.9Å of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr363 is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys146 positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr363 interaction with Lys146 and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases. Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys146 → Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys229 in native aldolase. Mutation of conserved active site residue Lys146 to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr363 hydroxyl within 2.9Å of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr363 is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys146 positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr363 interaction with Lys146 and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases. Stereospecificity is one of the hallmarks of enzyme catalysis. Aldolases, which are abundant and ubiquitous enzymes, catalyze stereospecific carbon-carbon bond formation, one of the most important transformations in living organisms. Their role is best known in glycolysis where fructose-1,6-bis(phosphate) (FBP) 3The abbreviations used are: FBP, fructose-1,6-bis(phosphate); G3P, d-glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; WT, recombinant wild type; DERA, d-2-deoxyribose-5-phosphate aldolase. aldolases (EC 4.1.2.13) promote the cleavage of FBP to triose phosphates, d-glyceraldehyde-3-phosphate (G3P), and dihydroxyacetone phosphate (DHAP). A common feature to class I enzymes is the use of a covalent mechanism for catalysis implicating iminium (protonated Schiff base) formation between a lysine residue on the enzyme and a ketose substrate (1Grazi E. Rowley P.T. Chang T. Tchola O. Horecker B.L. Biochem. Biophys. Res. Commun. 1962; 9: 38-43Crossref PubMed Scopus (68) Google Scholar) that entails stereospecific proton exchange in the covalent intermediate (2Rose I.A. Rieder S.V. J. Biol. Chem. 1958; 231: 315-329Abstract Full Text PDF PubMed Google Scholar). Of the three aldolase isozymes found in vertebrates (3Penhoet E.E. Rutter W.J. J. Biol. Chem. 1971; 246: 318-323Abstract Full Text PDF PubMed Google Scholar), the catalytic mechanism has been extensively studied using class I aldolase A from rabbit muscle and key intermediates are depicted in reaction Scheme 1. In the condensation direction, the reaction involves covalent intermediate formation with the keto triose phosphate DHAP followed by condensation with the aldehyde G3P to form the ketose of the acyclic FBP substrate (4Rose I.A. Warms J.V. Biochemistry. 1985; 24: 3952-3957Crossref PubMed Scopus (22) Google Scholar, 5Ray B.D. Harper E.T. Fife W.K. J. Am. Chem. Soc. 1983; 105: 3731-3732Crossref Scopus (15) Google Scholar). To form the C3-C4 bond of FBP, the enzyme stereospecifically abstracts the pro(S) C3 proton of the trigonal iminium 1 (6Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology, pp. 120-121, McGraw-Hill Book Co., New YorkGoogle Scholar, 7Grazi E. Cheng T. Horecker B.L. Biochem. Biophys. Res. Commun. 1962; 7: 250-253Crossref PubMed Scopus (72) Google Scholar) that is formed from the Michaelis complex with DHAP thereby generating via the enamine 2 (2Rose I.A. Rieder S.V. J. Biol. Chem. 1958; 231: 315-329Abstract Full Text PDF PubMed Google Scholar) the carbanionic character at C3 of DHAP for the aldol reaction. The nascent carbon-carbon bond has the same orientation as the pro(S) α-hydrogen initially abstracted from the DHAP imine intermediate. The overall retention of configuration at C3 requires that proton abstraction from 1 to yield the enamine 2 and condensation with aldehyde in 3 must take place from the same direction on the enzyme (8Rose I.A. J. Am. Chem. Soc. 1958; 80: 5835-5836Crossref Scopus (64) Google Scholar). The iminium intermediate formed is then hydrolyzed and FBP is released by the inverse reaction sequence shown in Scheme 1. A distinguishing mechanistic feature of class I aldolases is the relative stability of the iminium 1 and enamine 2 forms, which is a consequence of the catalytic requirements. The enzyme must stabilize the enamine 2 and/or the preceding iminium 1 such that no decomposition occurs prior to reaction with the aldehyde as shown in 3. This stability is reflected in solution where the enzymatic populations 1 and 2 represent 20 and 60%, respectively, of bound DHAP on the muscle enzyme under equilibrium conditions (9Rose I.A. Warms J.V. Kuo D.J. J. Biol. Chem. 1987; 262: 692-701Abstract Full Text PDF PubMed Google Scholar). The interconversion between the two forms implicates the conserved C-terminal Tyr363 residue whose proteolysis inhibits the stereospecific proton exchange step, making it rate-limiting (10Rose I.A. O'Connell E.L. Mehler A.H. J. Biol. Chem. 1965; 240: 1758-1765Abstract Full Text PDF PubMed Google Scholar), whereas the penultimate residues (357-362) of the C-terminal region modulate the rate of exchange reaction (11Berthiaume L. Tolan D.R. Sygusch J. J. Biol. Chem. 1993; 268: 10826-10835Abstract Full Text PDF PubMed Google Scholar). The C-terminal region (residues 343-363) is conformationally mobile (12Adelman R.C. Morse D.E. Chan W. Horecker B.L. Arch. Biochem. Biophys. 1968; 126: 343-352Crossref PubMed Scopus (26) Google Scholar, 13Sygusch J. Beaudy D. Allaire M. Arch. Biochem. Biophys. 1990; 283: 227-233Crossref PubMed Scopus (8) Google Scholar), has an extended secondary structure (14Blom N. Sygusch J. Nat. Struct. Biol. 1997; 4: 36-39Crossref PubMed Scopus Google Scholar, M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and aldolases and a of structural been that intermediates in class I aldolases M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Tolan D.R. Biochemistry. PubMed Scopus Google Scholar, E. E. P. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. I.A. PubMed Scopus Google Scholar), of the interconversion between the iminium and the enamine has at the structural To investigate the mechanism of proton in class I aldolases and the enzyme is to use of intermediates that are high resolution were of rabbit muscle class I FBP aldolase in complex with DHAP using aldolase in the presence of DHAP in to (9Rose I.A. Warms J.V. Kuo D.J. J. Biol. Chem. 1987; 262: 692-701Abstract Full Text PDF PubMed Google Scholar). of a native rabbit muscle aldolase crystal in a DHAP solution the enamine intermediate. the a crystal of the active site mutant Lys146 → Met revealed DHAP bound as the iminium intermediate and with the C-terminal in the active Attachment by the C-terminal region reaction for incipient proton at the DHAP C3 carbon and proton exchange and and of recombinant native and Lys146 → Met mutant rabbit muscle aldolases were as (11Berthiaume L. Tolan D.R. Sygusch J. J. Biol. Chem. 1993; 268: 10826-10835Abstract Full Text PDF PubMed Google Scholar, M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Tolan D.R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and using for of recombinant using an of at T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and aldolases were using the conditions M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). pro(S) proton exchange at the C3 carbon of DHAP by and aldolases and aldolase followed by of the as from of the at of DHAP in the presence of using an exchange in the (8Rose I.A. J. Am. Chem. Soc. 1958; 80: 5835-5836Crossref Scopus (64) Google Scholar). aldolase to conditions (10Rose I.A. O'Connell E.L. Mehler A.H. J. Biol. Chem. 1965; 240: 1758-1765Abstract Full Text PDF PubMed Google Scholar). of aldolase by A by of enzymatic activity using a and at E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The enzyme from A by aldolase activity decreased to of the and aldolase were for 2 in DHAP 1 in phosphate for 20 A mutant aldolase crystal for in a a of DHAP 20 to were in a and in a of to were at of the and were using a were with W. 1997; PubMed Scopus Google Scholar) and the are in and in are for the resolution in are for the resolution in are for the resolution with the of of of where is a from the observed prior to used and and of the for and by in are for the resolution with the of of where is a from the observed prior to used and and of the for and by J. 1993; Google Scholar). in a and crystal structures were with the crystal structure of native aldolase and to the were solved by using a native aldolase structure as M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). one consistent with the active form of the using an were to the resolution in the 1 to at in the resolution with an as M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) using the and P. J. N. T. Biol. PubMed Scopus Google Scholar) and M. A PubMed Scopus Google Scholar). The used to and Biol. PubMed Scopus Google Scholar). The presence of in the by of with and J. 1993; Google Scholar), are shown in 1. The and structure of aldolase with DHAP, aldolase with phosphate and aldolase with DHAP been with the and The structure of and The in using were at and in bond and are as and were on in of the aldolase were using the The Scholar). were with the of aldolase residues that are to binding as M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of covalent intermediates in the and structures from using the in The the of Lys229 and and DHAP and in enamine and iminium forms to the In the Lys229 whereas in the the The of in between the iminium and the enamine using a for identical the bound DHAP as iminium enamine of a aldolase crystal in the presence of DHAP a covalent intermediate in aldolase Lys229 in shown in formation of a covalent with The of the observed the DHAP carbon trigonal whereas of the the Lys229 and is consistent with trapping of a intermediate in aldolase of between bound DHAP and 3 and 3 respectively, active site by To the M. A PubMed Scopus Google Scholar) to to the The on for DHAP as an enamine an iminium with the enamine in This is and trapping of a enamine intermediate. exchange in aldolase as by of within the of DHAP the aldolase crystal and corroborates equilibrium trapping of an enamine intermediate in of DHAP and a structural feature in the enamine of the intermediate as the of enzymatic intermediates formed with DHAP in aldolase active of enzymatic intermediates used to the of the an enamine intermediate in in the structure of native aldolase on the of of Lys229 and bond formation between the hydroxyl and Lys229 shown as in the to 1 and at an iminium intermediate in in the structure of the mutant aldolase with of Lys229 bond formation with from a Lys229 and DHAP and at pro(S) proton exchange at C3 of exchange using at step of the of used to used for rate in the absence of using at step of the of used to used for rate in the absence of aldolase. in a with active site residues stabilize the covalent as shown in 1. are identical to in the structure of the iminium intermediate formed with FBP substrate by the same enzyme M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) an bond with and Lys146 stabilize the intermediate by with DHAP C3 hydroxyl and on the of with the Lys146 is An bond between hydroxyl and Lys229 the enamine intermediate which is in the the iminium at Lys229 of the enamine by the bond is reflected in the pro(S) proton exchange rate at C3 of DHAP for to and and with native enzyme mechanism of the aldolase from rabbit of and from the active Scholar). of in a in exchange whereas the same bond in the rate site binding of DHAP identical in aldolase with to native enzyme that in the of the active site to the observed in the structure of the iminium intermediate formed with FBP M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of for the FBP bound are with residues of the which of the active site and by DHAP of for residues on of native enzyme using of residues of for residues on of native enzyme using of residues of for residues on of native enzyme using of residues of for residues on of native enzyme using of residues of for residues on of native enzyme using of residues M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of for residues on of native enzyme using of residues in a of the native and structures two conserved water and whose positions DHAP attachment that has a to that of of residues is by with and at from DHAP and C3 The orientation of is to the enamine and in with the DHAP A in with the C3 carbon a by the water binding to aldolase is by DHAP Mehler A.H. Biochemistry. PubMed Scopus Google Scholar) for the phosphate binding To the of the in stabilization of the enamine a aldolase crystal in a phosphate analysis revealed in to phosphate binding with to the of of the active site and binding by active site attachment of C-terminal residues 360-363. In one phosphate binding with the DHAP phosphate site in the enamine structure and with the C terminus the active site The by phosphate bound at the phosphate binding site were in and with by DHAP with to the native In from the region by the to with Tyr363 in the active An phosphate binding site found at the and with residues and the from an In two and the phosphate binding site is from the DHAP phosphate binding the of the active site at site active site attachment by the C terminus region and is of as phosphate positions to The C-terminal are bound at as in the enamine structure and C terminus Tyr363 with the same residues in phosphate binding as at the of D. In the in the active site as a of the two phosphate binding which of the C-terminal consistent with the for C terminus to A in the presence of phosphate (12Adelman R.C. Morse D.E. Chan W. Horecker B.L. Arch. Biochem. Biophys. 1968; 126: 343-352Crossref PubMed Scopus (26) Google Scholar) that of phosphate active site of as observed in and D. of Lys146 to Met aldolase catalytic activity Tolan D.R. Biochemistry. PubMed Scopus (68) Google Scholar) and stereospecific proton exchange at the shown in of a mutant crystal in the presence of DHAP a covalent intermediate in the active site of The by DHAP to the shown in and as an iminium of an enamine a of of between bound DHAP and 2 and 3 respectively, active site by attachment by DHAP a of Lys229 DHAP and as as an bond with the bound at the same in a structure with the and J. that the active site with to binding at the phosphate binding in of the the residues of the C-terminal in the native and enamine were bound in the active site with Tyr363 to the iminium intermediate of the bound C-terminal region with that of the same C-terminal region observed in of for of residues that the observed C-terminal is with active site shown in that are conserved Biol. PubMed Scopus Google Scholar). two of one of which with the Tyr363 observed in the whereas in the a with in site interaction positions the Tyr363 hydroxyl from the DHAP C3 carbon and to the re face of the as by DHAP carbon site attachment by the C terminus an of the Lys146 → Met as C terminus binding observed in the active site of mutant aldolase. and J. DHAP binding the same in as in the enamine of the residues were in of the The by the of the interaction between and that the residues C-terminal to of the C terminus the active the structures of and were the native were for aldolase and are a of the C-terminal an observed in the where the C terminus is bound in the active site and a observed in of the and structures 3 and where the C-terminal region the active C-terminal active site Tyr363 with and with and with which in with The attachment by with the In the the the active site (residues and with the C-terminal region the active site by relative to the structure of the enamine. The of the a by two water that involves The in the entails with and and with a water molecule in and water molecule which in hydroxyl C terminus and and of the to with the C-terminal by of the is to an with Tyr363 The bond between Tyr363 and Lys146 in the structure in a of between the Tyr363 hydroxyl and DHAP C3 carbon the enamine structure The is in the iminium structure by of Tyr363 and site by the Tyr363 hydroxyl in the iminium the conserved water molecule bound by in of and The to the enamine and iminium intermediates of the aldolase catalytic in the a to with proton during of the enabled a of the stereospecific proton exchange by the C-terminal region in class I DHAP covalent enzymatic in the presence of DHAP in of native and mutant rabbit muscle aldolases represent enamine and iminium A feature of the enamine structure is hydroxyl Lys229 that is in the enamine form and in the iminium of the enamine intermediate is by and which bond DHAP and the DHAP with to the and C3 that is for stabilization of the enamine The bond between and the DHAP phosphate in iminium and enamine is in the of the phosphate attachment by DHAP which is by bond formation J. Biochemistry. 1997; PubMed Scopus Google Scholar). C-terminal trapping of the C terminus in the active site of the shown in is the structural that C-terminal Tyr363 in stereospecific proton exchange in rabbit muscle aldolase (10Rose I.A. O'Connell E.L. Mehler A.H. J. Biol. Chem. 1965; 240: 1758-1765Abstract Full Text PDF PubMed Google Scholar). active site binding as a of the is as an identical interaction with active site residues is observed in the by the C-terminal region in the mutant iminium intermediate that the to C-terminal binding is by binding with active site residues and to of water and of the two DHAP bound structures that in the enamine active site and charge of by interaction with the Tyr363 the active site interaction of interaction transient C-terminal attachment that is in with the absence of C-terminal binding in the native enamine by the C-terminal region is for catalytic as in the the C terminus with the nascent G3P of the FBP as in interaction by the C-terminal region active site and/or substrate The of the C-terminal region and active site are mechanistic the C-terminal region as a mobile by the C-terminal region with active site residues are to of active site by C-terminal of class I aldolase The penultimate residues of the C-terminal sequence are conserved and and with the active site of and conserved The of the residues sequence of the penultimate residues with to catalytic in active site binding to sequence at residues and is consistent with of the DHAP exchange reaction by implicating penultimate residues of the C-terminal region in aldolase (11Berthiaume L. Tolan D.R. Sygusch J. J. Biol. Chem. 1993; 268: 10826-10835Abstract Full Text PDF PubMed Google Scholar). structures are consistent with Tyr363 the re face stereospecific pro(S) proton exchange with to the DHAP C3 carbon in the iminium and enamine intermediates of rabbit muscle aldolase. The of Tyr363 in the active site is of residues and a water molecule that for a solution for Tyr363 and is by a for the in abstraction in muscle aldolase I.A. Biochemistry. PubMed Scopus Google Scholar). A has been for the at DHAP C3 J. Am. Chem. Soc. Scopus Google Scholar), which in the iminium to a that of a J. Am. Chem. Soc. Scopus Google Scholar, J. J. Am. Chem. Soc. PubMed Scopus Google Scholar, Chem. Biol. PubMed Scopus Google Scholar), proton by of the and structures the structure M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) positions the C terminus Tyr363 hydroxyl in with the of the FBP molecule a common to bond formation at C3 in the proton exchange and G3P and overall retention of configuration at C3 in the enamine (8Rose I.A. J. Am. Chem. Soc. 1958; 80: 5835-5836Crossref Scopus (64) Google Scholar). A catalytic mechanism is in Scheme 2 the and and FBP condensation in class I FBP interconversion between the iminium and enamine The catalytic by DHAP binding the active site to stabilize iminium formation as M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), shown in Scheme The hydrolyzed by the conserved water molecule activated by and is to the by DHAP carbon and Lys229 and the DHAP carbon of the reaction binding by reaction in and Tyr363 hydroxyl is activated by proton the conserved water molecule to the iminium phosphate that as a reaction abstraction the Tyr363 that is stabilized by consistent with interaction in the stereospecific pro(S) abstraction by Tyr363 in reaction the C terminus is followed by of water molecule The enamine phosphate proton via water and to the form of for aldehyde in reaction attachment of G3P and the character at of the activated G3P aldehyde The form of bond formation, shown in reaction the FBP iminium in M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). during the catalytic is consistent with a of interconversion of the iminium to the enamine that entails by the Tyr363 in the active Scheme reaction in to the interconversion mechanism is the of the Tyr363 has been in proton in the catalytic mechanism of the aldolase M. J. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. D. Sygusch J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and as a the proton of the Tyr363 is as it of the Tyr363 hydroxyl the active site for with which is the of the Tyr363 hydroxyl from a by the bond to a by with the water molecule to the DHAP with DHAP C3 the in bond formation with Lys146 and the Tyr363 The phosphate E. Biophys. PubMed Scopus Google Scholar) the role of the phosphate as the at proton the water molecule The mechanism explains the absence of exchange in the presence of dihydroxyacetone binding in iminium enamine formation E. Biochemistry. PubMed Scopus Google Scholar) as the is to catalyze proton in the phosphate has been shown to catalyze whereas the the same D.J. J. Am. Chem. Soc. Scopus (26) Google Scholar). The catalytic implicating the C terminus in class I aldolase catalysis (10Rose I.A. O'Connell E.L. Mehler A.H. J. Biol. Chem. 1965; 240: 1758-1765Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The rate Tyr363 by proteolysis W.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) is consistent with water molecule activated by shown in between iminium to a pro(S) The activated water molecule is for at the DHAP carbon, whereas a the DHAP C3 carbon in with Lys146 and it to catalyze the pro(S) transfer. stabilization of a hydroxyl by and of the for and a of enamine formation on the of lysine analysis of enamine formation in the enzyme a in DHAP as enamine from to and shown to on a of two with of I.A. Biochemistry. PubMed Scopus Google Scholar). C-terminal C-terminal is conserved in class I FBP aldolases to found in as as Biol. PubMed Scopus Google Scholar). proteolysis of the C-terminal region inhibits exchange in the the enzyme activity as proteolysis the to exchange FBP with G3P (10Rose I.A. O'Connell E.L. Mehler A.H. J. Biol. Chem. 1965; 240: 1758-1765Abstract Full Text PDF PubMed Google Scholar, I.A. O'Connell E.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the same and active site of catalytic residues as class I aldolases J. Tolan D.R. Biochemistry. PubMed Scopus Google Scholar) by the stability of enamine E. J. Biol. Chem. Full Text PDF PubMed Google Scholar) to an absence of a mechanistic interconversion of the enamine to the in to class I the active site of has no residue of charge stabilization to the C3 carbon of dihydroxyacetone and the enzyme a mobile C-terminal region of J. 1997; PubMed Scopus (64) Google Scholar). In the proton exchange mechanism interconversion of the iminium to enamine in d-2-deoxyribose-5-phosphate aldolase a water molecule that is activated by to the iminium forming on the of as for stereospecific pro(S) of the carbon M. I.A. PubMed Scopus Google Scholar). The water molecule is for proton at the in the enamine and as in class I FBP iminium at the cleavage activity of native that of muscle aldolase by DERA, to muscle has a C-terminal region and of C terminus to catalytic activity by M. I.A. PubMed Scopus Google Scholar). the residues of the C-terminal region in are conformationally that the C terminus the active site M. I.A. PubMed Scopus Google Scholar). site by C terminus to promote exchange during as in muscle the mechanistic of the same water molecule in two it is that in FBP which a mobile C terminus the cleavage reaction rate is 2 of with FBP aldolases J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We for proton exchange with the aldolase and and for and by L. We at site

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