Ambiguities in Mapping the Active Site of a Conformationally Dynamic Enzyme by Directed Mutation

Abstract

On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species. Purified mutants (Asp21 → Ala, Asn38 → Ala, Asn38 → Asp, Asn38 → Glu, Thr42→ Ala, and Arg419 → Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation. Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lowerk cat 20- and 10-fold, respectively. Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increasesK m for all substrates. Asn38hydrogen-bonds directly to the 5′-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect onK m values, but reduces k catby 100-fold. The mutation Asn38 → Asp causes 10–57-fold increases in K m for all substrates along with a 30-fold decrease in k cat. At pH 5.6, however, the Asn38 → Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme. Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here. The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme. On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species. Purified mutants (Asp21 → Ala, Asn38 → Ala, Asn38 → Asp, Asn38 → Glu, Thr42→ Ala, and Arg419 → Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation. Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lowerk cat 20- and 10-fold, respectively. Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increasesK m for all substrates. Asn38hydrogen-bonds directly to the 5′-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect onK m values, but reduces k catby 100-fold. The mutation Asn38 → Asp causes 10–57-fold increases in K m for all substrates along with a 30-fold decrease in k cat. At pH 5.6, however, the Asn38 → Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme. Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here. The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme. Adenylosuccinate synthetase (IMP:l-aspartate ligase (GDP-forming); EC 6.3.4.4, AMPSase) 1The abbreviation used is: AMPSase, adenylosuccinate synthetase. 1The abbreviation used is: AMPSase, adenylosuccinate synthetase. is an essential enzyme in most organisms (for review, see Ref. 1Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell Regul. 1983; 22: 103-141Crossref PubMed Scopus (79) Google Scholar), catalyzing the first committed step in the biosynthesis of AMP. IMP+L­aspartate+GTP↔adenylosuccinate+GDP+phosphate REACTION1Primary sequences of AMPSase (2Wolfe S.A. Smith J.M. J. Biol. Chem. 1988; 263: 19147-19153Abstract Full Text PDF PubMed Google Scholar, 3Wiesmuller L. Wittbrot J. Noegel A. Schleicher M. J. Biol. Chem. 1991; 266: 2480-2485Abstract Full Text PDF PubMed Google Scholar, 4Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar, 5Mäntsälä P. Zalkin H. J. Bacteriol. 1992; 174: 1883-1890Crossref PubMed Google Scholar, 6Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar, 7Schabes A.V. Andreichuck Y.V. Holmes W.M. Domkin V.D. J. Biol. Chem. 1993; 268: 20191-20197PubMed Google Scholar, 8Guicherit O.M. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 9Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomv J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Fuhrmann J.L. Geoghagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4623) Google Scholar, 10Bouyoub A. Barbier G. Forterre P. Labedan B. J. Mol. Biol. 1996; 261: 144-154Crossref PubMed Scopus (17) Google Scholar) are 40% identical for any pairwise comparison, indicating a strong tendency to preserve a primordial gene throughout evolution (10Bouyoub A. Barbier G. Forterre P. Labedan B. J. Mol. Biol. 1996; 261: 144-154Crossref PubMed Scopus (17) Google Scholar). The enzyme putatively facilitates the formation of 6-phosphoryl-IMP by the nucleophilic attack of the 6-oxo group of IMP on γ-phosphate of GTP, and then the formation of adenylosuccinate by displacement of the 6-phosphoryl group by l-aspartate (11Lieberman I. J. Biol. Chem. 1956; 223: 327-339Abstract Full Text PDF PubMed Google Scholar, 12Fromm H.J. Biochim. Biophys. Acta. 1956; 29: 255-262Crossref Scopus (26) Google Scholar). AMPSase from Escherichia coli is a monomer at physiological concentrations (1 μm), but dimerizes when nucleotide ligands are present (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 14Kang C. Kim S. Fromm H.J. J. Biol. Chem. 1996; 271: 29722-29728Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The dimer is probably the physiologically active form of the enzyme, having a K m corresponding to intracellular concentrations of IMP. The disordered active site of unligated AMPSase becomes ordered in the presence of substrates and substrate analogs (15Poland B.W Silva M.M. Serra M.A. Cho Y. Kim K.H. Harris E.M.S. Honzatko R.B. J. Biol. Chem. 1993; 268: 25334-25342Abstract Full Text PDF PubMed Google Scholar, 16Silva M.M. Poland B.W. Hoffman C.R. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1995; 254: 431-446Crossref PubMed Scopus (38) Google Scholar, 17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, 18Poland B.W. Hou Z. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1996; 271: 15407-15413Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 19Poland B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Poland B.W. Lee S. Subramanian M.V. Siehl D.L. Anderson R.J. Fromm H.J. Honzatko R.B. Biochemistry. 1996; 35: 15753-15759Crossref PubMed Scopus (40) Google Scholar). The largest conformational change is a 9-Å movement of the loop 42–53 (40s loop), which folds against the guanine nucleotide. Loop 120–131 (120s loop), which interacts with IMP, and loop 299–303 (300s loop), which interacts primarily with analogs of l-aspartate in crystal structures, become ordered in the presence of ligands. The conformational changes above putatively exemplify induced fit, a concept introduced by Koshland some three decades ago (21Koshland Jr., D.E. Boyer P.D. The Enzymes. I. Academic Press, New York1970: 341Google Scholar). The ligand-enzyme interactions, however, which contribute most (in terms of a thermodynamic driving force) to the observed conformational changes have yet to be identified, nor can we exclude the possibility of energy contributions from interactions between protein residues well removed from the active site. On the basis of ligated crystal structures of adenylosuccinate synthetase, Asp21, Asn38, Thr42, and Arg419 do not interact with atoms of substrates involved with the chemistry of phosphotransfer or nucleophilic attack by l-aspartate (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, 19Poland B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In fact, Asp21 and Arg419 do not interact with ligands (16Silva M.M. Poland B.W. Hoffman C.R. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1995; 254: 431-446Crossref PubMed Scopus (38) Google Scholar, 17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, 19Poland B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Poland B.W. Lee S. Subramanian M.V. Siehl D.L. Anderson R.J. Fromm H.J. Honzatko R.B. Biochemistry. 1996; 35: 15753-15759Crossref PubMed Scopus (40) Google Scholar). The backbone amide of Thr42 forms a hydrogen bond with the α-phosphoryl group of the guanine nucleotide, but its side chain forms only a weak hydrogen bond to that same α-phosphoryl group (20Poland B.W. Lee S. Subramanian M.V. Siehl D.L. Anderson R.J. Fromm H.J. Honzatko R.B. Biochemistry. 1996; 35: 15753-15759Crossref PubMed Scopus (40) Google Scholar). Asn38 provides one of five hydrogen bonds to the 5′-phosphoryl group of IMP; implicating Asn38 in the ground-state stabilization of the IMP-enzyme complex (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, 19Poland B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Nevertheless, mutations of Asn38 have little effect on theK m of IMP, but a major impact onk cat, mutations of Asp21 and Arg419 destabilize the transition state, and the mutation of Thr42 to alanine reduces affinities for all substrates. The effects of each of the mutations above can be understood in terms of their influence upon two interdependent, dynamic mechanisms in AMPsase: (i) ligand-induced reorganization of the active site and (ii) ligand-induced dimerization of the enzyme. The above mutations also illustrate the importance of a sound understanding of structure and dynamics of an enzyme before assigning functional roles to side chains. GTP, IMP, l-aspartate, phenylmethylsulfonyl fluoride, and bovine serum albumin were from Sigma. Restriction enzymes were from Promega. Pfu DNA polymerase and E. coli strain XL-1 blue were obtained from Stratagene. E. coli strain H1238 (purA−) was a gift from Dr. D. Bachman (Genetic Center, Yale University, New Haven, CT). Phenyl-Sepharose CL-4B came from Amersham Pharmacia Biotech. Other reagents and chemicals came from Sigma if not otherwise specified. Owing to the undetectable levels of expression for some mutants using the PMS204 plasmid expression system, we used here a more efficient prokaryotic expression system, based on the pTrc99A vector (Amersham Pharmacia Biotech). The purA gene was amplified by polymerase chain reaction using the following primers to incorporate NcoI andPstI restriction sites at either end of the purAgene. TrcN:TrcC2:5′­GATGCCATGG¯GTAACAACGTCGTCG­3′(NcoI site underlined)5′­AACTGCAG¯TCTGCCAGGCGTACCACA­3′(PstI site underlined) The NcoI restriction site was incorporated into the N terminus of the purA gene, before the first Met codon (ATG), and the PstI restriction site was introduced after the stop codon. Pfu DNA polymerase was used in the polymerase chain reaction to ensure high fidelity amplification (Fig. 1). The amplified and subcloned purA gene containing the 1.3-kilobase pair fragment was sequenced twice in both directions using the chain termination method (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) at the Nucleic Acid Facility, Iowa State University. Sequence analysis showed 100% identity with theE. coli purA sequence deposited in GenBank™, except at position 415, a GAT (Asp) is replaced by GGT (Gly), which agrees with published crystal structures (15Poland B.W Silva M.M. Serra M.A. Cho Y. Kim K.H. Harris E.M.S. Honzatko R.B. J. Biol. Chem. 1993; 268: 25334-25342Abstract Full Text PDF PubMed Google Scholar). The purA gene was subcloned into the pTrc99A vector and the final construct, pTrpA, was transformed into E. coli strain H1238 (purA−) for overexpression of AMPSase. TheNcoI-PstI fragment containing purA was subcloned in to the pAlterEXII vector (Promega, Inc.). Site-directed mutagenesis was carried out according to published procedures from Promega, Inc. The primers used in mutations were 5′-GTAAGATCGTCGCTCTTCTGACTGA-3′ (Asp21 → Ala), 5′-GGGCGGTCACGCTGCAGGCCATA-3′ (Asn38→ Ala), 5′-GGGCGGTCACGACGCAGGCCATA-3′ (Asn38→ Asp), 5′-GGGCGGTCACGAAGCAGGCCATA-3′ (Asn38 → Glu), 5′-CGCAGGCCATGCTCTCGTAATCAA-3′ (Thr42 → Ala), and 5′-CCGGATCTTACTGAAACCATG-3′ (Arg419 → Leu), where the underlined bases are mismatched with respect to the wild-typepurA sequence. The primer 5′-AACAACGTCGTCGTACTGGG-3′ was used to sequence the plasmids and confirm mutations for all but Arg419 → Leu, where the primer 5′-AACAGCCAAGCTTGCATGCC-3′ was used. All primers were synthesized on a Bioresearch 8570EX automated DNA synthesizer at the Nucleic Acid Facility at Iowa State University. After mutagenesis, the NcoI-PstI fragment containing the desired mutation was subcloned into pTrc99A and transformed into the E. coli strain H1238 (purA−) for expression. The wild-type and mutant enzymes were purified as described elsewhere (23Bass M.B. Fromm H.J. Stayton M.M. Arch. Biochem. Biophys. 1987; 256: 342-355Google Scholar, 24Kang C. Fromm H.J. Arch. Biochem. Biophys. 1994; 310: 475-480Crossref PubMed Scopus (14) Google Scholar, 25Wang W. Hou Z. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 16911-16916Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) with the following modifications. The enzyme was eluted from a phenyl-Sepharose CL-4B column by a series of buffered solutions, 0.6 m, 0.4 m, and 0.2m in (NH4)2SO4 and 50 mm in potassium Pi (pH 7.0). All the enzymes eluted at 0.2 m(NH4)2SO4. The enzyme fractions were concentrated and dialyzed against 50 mm potassium Pi (pH 7.0), then further purified using a DEAE-TSK high performance liquid chromatography column. Enzyme purity was monitored by SDS-polyacrylamide gel electrophoresis (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), and protein concentrations were determined using Bradford reagent (Bio-Rad) (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). All the mutant enzymes were subjected to circular dichroism analysis as described elsewhere (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar, 23Bass M.B. Fromm H.J. Stayton M.M. Arch. Biochem. Biophys. 1987; 256: 342-355Google Scholar, 24Kang C. Fromm H.J. Arch. Biochem. Biophys. 1994; 310: 475-480Crossref PubMed Scopus (14) Google Scholar). From 1 to enzyme was used in on the of each changes at and were monitored with a with a and were as for the pH and was used at pH concentrations were at mm for all GTP, mm for the wild-type enzyme and mm for the Asn38 → Asp mutant enzyme. IMP concentrations from to for of the wild-type enzyme, and to mm for of the Asn38 → Asp of the Asn38 → Asp mutant and 1 of wild-type AMPSase were used in were used to for the high IMP. was used to for the of the Asn38 → Asp Asn38 → Asp, and Asn38 → mutant were by equilibrium in the and in the presence of ligands mm IMP, GTP, 1 at pH and were as described (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), using in of Enzyme concentrations were that an of were determined as described (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). corresponding to Asp21, Asn38, Thr42, and Arg419 are 100% identical across In the of Asn38 has no interactions, but in the ligated synthetase with the of IMP (Fig. Ref. 17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). a (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, C. Poland B.W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), has three of (i) In the unligated synthetase, with M.M. Poland B.W. Hoffman C.R. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1995; 254: 431-446Crossref PubMed Scopus (38) Google Scholar, 17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). (ii) In the complex of IMP, and with the group of (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). In the complex of and with the group and to B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Asp21 in the unligated synthetase with as but also a with Arg419 in ligated (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). Thr42 has no in the unligated synthetase (16Silva M.M. Poland B.W. Hoffman C.R. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1995; 254: 431-446Crossref PubMed Scopus (38) Google Scholar, 17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar), but in ligated its backbone amide interacts with the α-phosphoryl group of and its backbone with a which in with the of and backbone In one of three ligated of the synthetase (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar, 19Poland B.W. Bruns C. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1997; 272: 15200-15205Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Poland B.W. Lee S. Subramanian M.V. Siehl D.L. Anderson R.J. Fromm H.J. Honzatko R.B. Biochemistry. 1996; 35: 15753-15759Crossref PubMed Scopus (40) Google Scholar), the side chain of Thr42 interact with the of to of Ref. 20Poland B.W. Lee S. Subramanian M.V. Siehl D.L. Anderson R.J. Fromm H.J. Honzatko R.B. Biochemistry. 1996; 35: 15753-15759Crossref PubMed Scopus (40) Google Scholar). The interactions above are in of E. coli AMPSase sequence and with the sequences of AMPSase from (2Wolfe S.A. Smith J.M. J. Biol. Chem. 1988; 263: 19147-19153Abstract Full Text PDF PubMed Google Scholar, 3Wiesmuller L. Wittbrot J. Noegel A. Schleicher M. J. Biol. Chem. 1991; 266: 2480-2485Abstract Full Text PDF PubMed Google Scholar, 4Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar, 5Mäntsälä P. Zalkin H. J. Bacteriol. 1992; 174: 1883-1890Crossref PubMed Google Scholar, 6Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar, 7Schabes A.V. Andreichuck Y.V. Holmes W.M. Domkin V.D. J. Biol. Chem. 1993; 268: 20191-20197PubMed Google Scholar, 8Guicherit O.M. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 9Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomv J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Fuhrmann J.L. Geoghagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4623) Google Scholar, 10Bouyoub A. Barbier G. Forterre P. Labedan B. J. Mol. Biol. 1996; 261: 144-154Crossref PubMed Scopus (17) Google in a interactions in the and ligated of E. coli with of of or of amide of of a in a All have an monomer of and purity than as by SDS-polyacrylamide gel plasmids containing the mutations of the on with and indicating enzyme to the transformed The of Asp21 → Ala, Thr42 → Ala, Arg419 → Leu, and wild-type enzymes are identical from to indicating the of conformational change of The position mutants showed in their to the wild-type not The are probably a of a in the of the loop the equilibrium of m of the Asp21 → Ala, Asn38 → Ala, and Asn38 → mutants are to of wild-type AMPSase cat for the Thr42 → mutant was the same as that of the wild-type enzyme, K showed The mutation of alanine k cat and and k cat 10-fold, with a in and increases in and Asn38 → Ala, Asn38 → Asp, and Asn38 → mutants showed in k to the wild-type enzyme. the three position Asn38 only Asn38 → Asp K m of wild-type and mutant → → → → → → Ref. → From Ref. in the in a the three position m for the Asn38 → Asp mutant for the Asn38 → Asp mutant from between the 5′-phosphoryl group of IMP and the side chain of of the side or the 5′-phosphoryl group of IMP, cat and K m to wild-type k cat pH for the wild-type and the Asn38 → Asp enzymes are (Fig. The pH for wild-type and Asn38 → Asp enzymes are and 5.6, respectively. In fact, the Asn38 → Asp mutant than the wild-type enzyme at pH and not wild-type levels of were in the Asn38 → Asp mutant by the 100-fold than that of the wild-type enzyme. IMP dimerization of (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 14Kang C. Kim S. Fromm H.J. J. Biol. Chem. 1996; 271: 29722-29728Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). the dimer has 100-fold for IMP than the we the Asn38 → Asp mutant by in to its from its properties of In reported the wild-type and Asn38 → enzymes are as each K m for IMP, and be in the presence of IMP. The for the three enzymes in the and presence of ligands at pH are in At pH 5.6, the three enzymes The three K in the of ligands. In the presence of however, the K the Asn38 → Asp mutant significantly from that of wild-type and Asn38 → both of which are the for Asn38 → Asp in the presence of ligands is than the for Asn38 → Asp in the of for wild-type and mutant of of of of determined by the of of determined by the → → of determined by the in the Scholar). in a the equilibrium mechanism of AMPSase M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell Regul. 1983; 22: 103-141Crossref PubMed Scopus (79) Google cat the of the complex of enzyme and substrates to enzyme and cat for is to the energy changes in the transition of the transition for the Asp21 → and Arg419 → mutants cat 20- and 10-fold, to the wild-type the the transition the hydrogen bond between Asp21 and Arg419 is from the of the stabilization is a of an The of the destabilize interactions between the and in the unligated enzyme Arg419 is disordered (15Poland B.W Silva M.M. Serra M.A. Cho Y. Kim K.H. Harris E.M.S. Honzatko R.B. J. Biol. Chem. 1993; 268: 25334-25342Abstract Full Text PDF PubMed Google Scholar, 16Silva M.M. Poland B.W. Hoffman C.R. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1995; 254: 431-446Crossref PubMed Scopus (38) Google Scholar) and the and loop do not In the ligated enzyme, backbone with a which in to backbone and the of (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). mechanism by which the of the influence the transition is by of a of the is a (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google C. Poland B.W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). displacement of its side chain by as little as to of the synthetase. The of Asp21 with Arg419 the position of which on the side of the in the have only effects on and no effect on theK m for substrates C. Poland B.W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), with the of change in K m for the Asp21 → The mutation of Arg419 to has a effect on theK m for IMP and The be a of a change in the loop as the Asp21 mutation has no effect on K m The mutation of Arg419 the of the guanine and to the in the observed of IMP and (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), a IMP The side chain of Thr42 binds to the α-phosphoryl group of GTP, yet is a conserved and its mutation to alanine increases K m by The above be a of the which is in an equilibrium between two conformational The in the of the when ligands are to the active site. The of an effect onk cat by the mutation of alanine no of the loop in the ligated of the synthetase. however, the mutation to alanine the of the then ligands a of their energy to the loop to its the K m for all substrates is a of in substrate (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Asn38 → and Asn38 → have no influence on K m but cat by and respectively. the between Asn38 and the of IMP not the of IMP for the active site. the energy probably is to the stabilization of the transition Asn38 to a that the the between residues and a change in (17Poland B., W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar) and the and of Asn38 the by 1 conformational the loop are probably to the 9-Å movement of the loop the energy of of Asn38 with the of IMP to the conformational change in the The mutation of Asn38 to a more complex The of a at position probably by All of the of its side chain to the of IMP. of are which of the and the side of the of IMP, a hydrogen bond is in the Asn38 → Asp k cat for the mutant at pH is to wild-type IMP can the conformational change in the K m for the Asn38 → Asp mutant are not to wild-type levels at however, that a mechanism is for the m of the The wild-type enzyme is a monomer in the of ligands at concentrations used in (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). IMP dimerization of the wild-type enzyme at pH its with of a monomer essential in the The mutation of to or not cat, but increases 100-fold at pH and ligand-induced dimerization at concentrations of IMP and enzyme (13Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The high for the Asn38 → Asp mutant at pH then the weak of IMP with the mutant as a than the of IMP to a mutant is by the for Asn38 → Asp with and ligands. The for the Asn38 → Asp in the presence to the of ligands that Asn38 → Asp a in the of ligands. At pH 5.6, IMP to the active site of the mutant as a formation of the hydrogen bond with the side chain of the of IMP not its with of a the for IMP the mutant a monomer at The wild-type enzyme, on the the of IMP from at pH as a the for wild-type enzyme at pH the of IMP with a synthetase is with ligand-induced which in a influence subunit dimerization reorganization of its active site. a of the of energy to dynamic that influence the of from the active site, residues can directly to substrates and have no effect on as by K m and residues from the active site can have effects on the of the transition or the of substrates. underscore the in using site directed mutations alone in assigning residues to the active site of an enzyme.