Mutagenic analysis of AMP nucleosidase from Escherichia coli. Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry.

AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K(+)-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD(+)-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.

AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, POr. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, r w proteins, RNA polymerase, K+-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD+-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was overproduced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in V,,, but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.
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Both prokaryotes and eukaryotes contain enzymes which regulate the cellular adenine nucleotide pool by the hydrolytic degradation of AMP. In prokaryotes, degradation occurs through hydrolysis of the N-glycosidic bond to yield adenine and ribose 5-phosphate (Schramm and Leung, 1978a). In eukaryotes, AMP deaminase carries out a similar function by deaminating AMP to IMP and ammonia (Chapman and Atkinson, 1973). No prokaryotes are known to contain AMP deaminase and no eukaryotes are known to contain AMP nucleosidase. Regulatory features of AMP nucleosidase and AMP deaminase are remarkably similar. Both are allosterically activated by ATP, and both are inhibited by inorganic phosphate (Schramm, 1974;Merkler et al., 1989). The hypothesis that AMP nucleosidase evolved into AMP deaminase as prokaryotes evolved into eukaryotes has been tested by cloning and sequencing the genes for these enzymes from Escherichia coli and from Saccharomyces cereuisiae (Leung et al., 1989;Meyer et al., 1989). Neither of the deduced sequences contained the consensus sequence for adenylate-binding proteins. The deduced amino acid sequences indicate only a small region of homology. Since both AMP nucleosidase and AMP deaminase contain ATPand AMP-binding sites, the absence of adenylate consensus sequences is surprising and requires verification. The common feature between these proteins is a small region of eight amino acids which is similar to peptides known to be involved in nucleotide or phosphate binding in other proteins including a well characterized domain of glyceraldehyde-3-phosphate dehydrogenase (Rossman et al., 1975).
In this article, the common sequence between AMP deaminase and AMP nucleosidase has been removed by deletion mutagenesis. The mutant protein has been produced and characterized in order to provide information on the nature of the sequence which is common to deaminase, nucleosidase, and other proteins which are known to have nucleotide-or phosphate-binding regions. The results establish that a deletion of eight amino acids changes primarily the catalytic efficiency and the polymeric state of the enzyme. The deduced structure of both the mutant and native AMP nucleosidase is experimentally verified by mass spectral analysis of peptides produced by proteolysis. This procedure ensures that the amino acid composition deduced from DNA sequence reflects the actual amino acid sequence. The mutated amn DNA from the M13 subclone marked in black was ligated to the native amn gene at the HindIII-PstI sites. The start ( I ) and the end (1450) of the amn gene are marked. The double lines represent the 2.65-kb HinfI fragment restricted initially from pHL8 and subcloned into M13mp18 . The single line represents pTZ18U.
Sephacel were obtained from Pharmacia LKB Biotechnologies. L-1-Tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin, Staphylococcus aureus VS protease,' all protease inhibitors, and lysozyme were from Sigma. Oligonucleotides for the deletion mutagenesis and DNA sequencing were produced by the DNA synthesis facility, Albert Einstein College of Medicine using Applied Biosgstems models 380 A and B DNA synthesizers. Acetonitrile was obtained from Burdick and Jackson Company. Glycerol was obtained from Fisher, and trifluoroacetic acid was obtained from Aldrich. All other chemi- a Purification was similar to the published method for native enzyme (Schramm and Leung, 1978b).
For further purification, A(128-135)AMN (20 mg) after chromatography on DEAE-Sephacel was subjected to fast protein liquid chromatography on Mono Q (HR5/5) in 100 mM Tris buffer, pH 8.0, containing 2 mM AMP. The purified enzyme was eluted with a 160 ml linear gradient of 0-0.5 M NaCl in 100 mM Tris buffer, pH 8.0, containing 2 mM AMP. cals were of reagent grade from various sources. The mutant AMP nucleosidase A(128-135)AMN was purified from extracts of HL359[pTZ-A(382-405)]amn (Leung et al., 1989). The purification procedure was simplified by overexpression to approximately 30% of the total protein as the mutant AMP nucleosidase. The purification procedure is summarized in Table I.
Mutagenesis-The 2.65-kb HinfI fragment of pHL8  was subcloned into the SmaI site of M13mp19 and used to generate the single-stranded amn template for mutagenesis. The single-stranded DNA was purified according to Davis et al. (1986). An oligonucleotide (5-CCT TAT GTC ATC CGC TCA ATG AGC GC) was designed to anneal to ann, loop out bases  in the amn gene, and to alter the ClaI restriction site. The deletion mutagenesis of amn with the HPLC purified oligonucleotide was performed according to the instructions provided by Amersham Corp.
Oligonucleotides and DNA Sequencing-The oligonucleotides for the sequencing of amn and the mutagenesis study were treated and purified as described previously (Leung et al., 1989). The DNA sequence of the single-stranded a n n gene after mutagenesis was obtained by the dideoxy sequencing method of Sanger et al. (1977), as adapted for 35S (Biggin et al., 1983) using Sequenase (T7 DNA polymerase). Restriction, ligations, and associated manipulations of DNA were according to the procedures in Maniatis et al. (1982).
Protease Digestion of AMN and A(128-1351AMN-The purified proteins (8 mg of AMN and 5 mg of A(128-135)AMN) were denatured in 7 M guanidine hydrochloride for 2 h at 37 "C. The proteins were dialyzed for 24 h at 4 "C against three changes of a 100-fold excess of 100 mM NH4HC03 buffer, pH 8.0, or 25 mM NH4HC03 buffer, pH 7.8, for treatment with trypsin or V8 protease, respectively. Trypsin or Va protease was dissolved a t 1 mg/ml in distilled water and was added in 4 aliquots over an 8 h period to equal 4% of the AMN proteins for trypsin or 5% of the AMN proteins for V8 protease. After incubation at 37 "C for 24 h, the samples were centrifuged to remove precipitate, lyophilized to dryness, and resuspended in distilled water at 1-5 mg/ml. One-half of each VS protease-treated sample was resuspended in 100 mM NH4HC03 buffer, pH 8.0. Trypsin was added to the samples to a final concentration of 4% trypsin to AMN protein.
In separate experiments, trypsin incubation controls did not give peaks which interfered with the analysis. The samples were clarified and concentrated as described above.

G S Y T T T I T R P~L T L L~~S V Q P S Q H E
" " " , 6 0 : 6 1 6 2

I D M E s A T P~L E G E~~ 440
1-72-73-6 3 w 6 4 9 6 5 n 6 8 F6_7+$8 ; 6 9 ; ;71+72-+-- Sample desorption and ionization was accomplished with an 8-keV neutral Xenon beam from an Ion-Tech saddle-field FAB source. Mass calibrations were performed with cesium iodide and glycerol. Scans were acquired over 15 s from 450 to 2200 atomic mass units using a Finnigan-MAT 90 at a source temperature of 55 "C, a resolution of 1400, and an accelerating voltage of 5 kV.

N R F Y E G A I -I G f R
Initial Rate Kinetic Studies-Initial reaction rates were measured at 30 "C in 1-ml reaction mixtures containing 0.1 M triethanolamine-HC1, pH 8.0, and the indicated concentrations of AMP and ATP. The concentration of MgC12 was equal to or greater than the ATP concentration to convert ATP to MgATP and to maintain free MgC12 between 10 p~ and 1 mM. The reaction was initiated by the addition of 5 pl of enzyme which had been diluted into 0.5 M triethanolamine, pH 8.0. Reactions were terminated by the addition of 0.3 ml of alkaline Cu2' reagent (Schramm and Hochstein, 1971) or by 20 pM formycin 5-phosphate, an inhibitor of the enzyme (DeWolf et al., 1979).
Assays with A(128-135)AMN gave initial rates which lagged for 1 or more min followed by product formation as a linear function of time. Initial rates were calculated from the linear portion of the product formation plots. Three or more time points were used to estimate steady-state reaction rates. Kinetic constants were obtained from fits of the data to the appropriate equations (Cleland, 1977), where appropriate. Preincubation of the enzyme in dilute buffer solutions prior to assay did not eliminate the hysteresis.
Presteady-state Kinetic Studies-Rapid reaction kinetics were conducted in a Kin Tech rapid quenching instrument at 30 "C. All solutions were buffered at pH 8.0 with 0.1 M triethanolamine, pH 8.0. The enzyme syringe contained 20 p~ (1.04 mg/ml) native or mutant AMP nucleosidase and was rapidly mixed with an equal volume containing the indicated concentrations of substrate and allosteric activator. The mixing time of the instrument is approximately 2 ms. Substrate AMP was mixed with ["PIAMP to provide radioactive product. After the appropriate incubation period, the reaction (82 p1) was terminated by the addition of 82 or 190 pl of 1 N HCI. The product ribose 5-phosphate was isolated on small disposable columns of charcoal-cellulose eluted with 10 mM unlabeled ribose 5-phosphate (Parkin et al., 1984). Control experiments demonstrated that ["PI AMP from experiments containing no enzyme gave no 32P eluting from the columns.
indicate peptides not observed in the analysis of the proteins. All numbers above the peptides are for the combined Vs and trypsin protease ( V T peptides). The mass range of the analysis was from 450-2200 amn, therefore peptides with 1-3 amino acids are not detected. Peptides from incomplete digestion are indicated, e.g. VT2-VT3, 12-13 etc. Peptide 16m is unique to the mutant protein and excludes the boxed residues. , with a gradient program of 0-100% B in 30 min after an initial isocratic hold of 5 min at 0% E. The flow rate was 10 pl/min.

RESULTS
Preparation of A(128-135)AMP Nucleosidase-A deletion mutant protein of AMP nucleosidase [A(128-135)AMN] was prepared by oligonucleotide-directed mutagenesis, as described under "Experimental Procedures." The mutated double-stranded DNA from individual M13 isolates was screened by ClaI restriction analysis. Since the ClaI site in amn was deleted during the mutagenesis the desired mutations gave restriction fragments of 2.9 and 7 kb whereas native amn DNA gave three bands of 1.3, 2.9, and 5.7 kb. One-third of the M13 progeny were shown to have lost the amn ChI site.
The deoxyribonucleotides 1-500 surrounding the deletion of the mutated A(382-405)arnn gene were sequenced and compared with the sequence of the parental gene (Leung et al., 1989) (Fig. 1). The results verified that the deletion had occurred in the DNA. No other mutations were observed in deoxyribonucleotides 1-500 of the coding region of the amn gene. A pTZ-A(382-405)amn construct was prepared containing the promotor and bases (1-477) of the A(382-405)arnn gene. The mutant DNA was ligated to the remainder of the native amn gene at the HindIII-PstI sites (Fig. 2). The DNA sequence of bases -24 to 500 of the final construct confirmed the DNA structure. The pTZ-A(382-405)arnn gene was transformed into HL359, an E. coli strain deficient in native AMN activity  and devoid of AMN protein as shown by Western blot analysis.
The A(128-135)AMN protein was purified using a proce- 1021 in panel G is unique to the mutant protein and is not seen in the wild-type protein (C). Spectra obtained by accumulating the scans associated with the chromatographic peak were used to determine their m/z values and allow assignment to the appropriate peptide. The relative intensities in panels A , B, D, F, and G were normalized to the most intense peak signal. The relative intensity of panel C was normalized to that of panel G, while those of panels E and H were normalized to that of A and D, respectively. The retention times of peptides from both digests were within 2% of each other after 60 min of chromatography at 10 pl/min. Equal quantities of peptide digest were applied for analysis of AMN and mutant digests.

SCAN NUMBER
dure similar to the published method of Schramm and Leung (1978b) (Table I). Denaturing SDS gel electrophoresis of the protein at each step of the purification of A(128-135)AMN indicated that the mutant protein was expressed at >30% of total extracted protein and was purified to >99% homogeneity (Fig. 3). Protease Digestion of Native AMN and A(l28-135)AMN-Native AMP nucleosidase and mutated A(128-135)AMN were treated with Vs protease or trypsin, either separately or in combination. Incomplete protease digestion and/or recovery of peptides from both native and mutant proteins was evidenced by the presence of protein precipitates. Precipitates were removed by centrifugation prior to analysis by mass spectrometry. Trypsin digestion yields two large peptides of M, 4867 for AMN and 4036 for A(126-135)AMN in the region of the deletion. For FAB mass spectrometic analysis, smaller peptides were generated by combined digestion with trypsin and V8 protease.
Mass Spectral Analysis of Native and A(128-135)AMN Peptides-The product of Vs protease/trypsin (VT) digestion was analyzed using HPLC-MS. The spectra and chromatograms of similar analyses were compared for peptides from the wildtype protein and for the mutant. The peptides VT16 and VT17, comprising sequence positions 121-131 and 132-136 were predicted to appear only in digests of native AMN. A single peptide, VTlGm, spanning sequence positions 121-128 of the mutant protein, was predicted to be unique to the mutant protein, since it contains residues which span the deletion site (Fig. 4). Direct mass spectral analysis of the HPLC effluent was used to provide masses of all peptides within the range 450-2200 atomic mass units which were present in the digest. The  Fig. 7, in which the scans corresponding to some of the peaks in Fig. 6 were accumulated and the background was substracted. The upper spectra of Fig. 7 show that both VT17 (m/z 617.5) and VT16 ( m / z 1253.1) are present in the AMN digest, but not in the digest of the mutant protein (lower spectra). Peptide VT22 is expected and is found in both the native and mutant proteins (Fig. 7, B and E ) . The VT16 and VT17 peptides are replaced in the A( 128-135) AMN by peptide VT16m (m/z 1021.0) which is clearly detected as a major peak in the mutant but not the native digests (compare Fig. 7, C and F ) . Table I1 summarizes the results of the LCMS analysis of both proteins. The m/z values of the assigned peptides were generally within 0.5 mass units of the predicted values. The highest mass peptide observed was VT42 (mlz 2126.10) and was observed as a weak ion in the digest of the mutant protein.
Several ions were assigned to products of incomplete diges-tion, i.e. peptides which contain a lysine, glutamic acid, or arginine in their internal sequence. The peptides VT59 and VT60 were not expected because it would require the cleavage of a lysine-proline peptide bond. Such bonds are resistant to cleavage by trypsin. The ion at m/z 903 could be attributed to peptides VT26, VT34, or both. Of the peptides which required the cleavage of an Arg-Pro peptide bond, only VT12, VT13, and VT45 were observed, while peptides VT37, VT38, VT40, and VT41 were not observed in either digest.
Kinetic Properties of Native and A(l28-135)AMN"Initial rate studies with A(128-135)AMN gave a lag period of 1-2 min before a linear rate of product formation was observed when MgATP was present (Fig. 8A). This hysteresis was not observed with the native enzyme (Fig. 8C). In the absence of MgATP, the hysteresis was extended to 20 min or more, making initial rate measurements difficult (Fig. 8B). Preincubation of enzyme in buffer of lower ionic strength, with and without MgClz, AMP, formycin 5-phosphate, ATP, and MgATP did not eliminate the hysteresis, suggesting that multiple catalytic turnovers are required to form a stable catalytic unit. In stopped-flow studies, using 10 g M A(128-135)AMN or native AMN, no hysteresis was observed in the absence or presence of ATP (Fig. 9). The concentration of A(128-135)AMN was 0.02-0.2 ~L M in kinetic studies. The hysteresis is therefore consistent with a concentration-dependent protein polymerization which is linked to catalytic function. Initial rate measurements for A(128-135)AMN were taken after the lag was complete. Because of the relatively long delays under some conditions (Fig. 8), the initial rates are not highly reproducible.
The AMP saturation curve for native AMP nucleosidase in the absence of allosteric activator gave a half-saturation value of 15 mM. The estimated So.5 for AMP with A(128-135)AMN  was 23 mM using steady-state rates established after 30 min at 30 "C (see Fig. 8). Under these conditions, the native enzyme had a VmaX of 40 and the A(128-135)AMN had a VmaX of 0.42 bmol/min/mg. Both enzymes exhibited sigmoidal responses of initial rates to AMP concentration.
Addition of MgATP caused a decrease in the values for substrate to 90 and 210 PM for the native and mutant enzymes and caused substrate-saturation to conform to Michaelis-Menten kinetics. The V,,, value was unchanged for the native enzyme but increased &fold for A(128-135)AMN in response  Table 111. Activation by MgATP gave a sigmoidal response of initial rate to activator concentration when AMP was fixed at concentrations below 1 mM. The apparent activation constants for MgATP were 22 and 100 p~ for native and A(128-135)AMN.
Inhibition of AMP nucleosidases occurs with inorganic phosphate. With equivalent fixed concentrations of AMP and MgATP, the native and A(128-135)AMN gave apparent inhibition constants of 200-250 p~ (Table 111). Inhibition occurs in the presence and absence of MgATP, but the observed inhibition constants are greater in the presence of MgATP.
Formycin 5-phosphate is a competitive inhibitor of native AMP nucleosidase and also inhibits A(128-135)AMN. The inhibition constants were estimated from titrations of activity as a function of formycin 5-phosphate with fixed concentrations of AMP.

136)AMP nucleosidase ( B ) .
Native AMP nucleosidase was mixed with substrate to give final reaction conditions of 10 p~ catalytic sites, 0.5 mM MgATP, 150 @M AMP, and 0.5 mM MgC12. AMP nucleosidase A(128-135) was mixed with a substrate plus MgATP solution to give a final reaction mixture of 10 fiM catalytic sites, 0.5 mM MgATP, 0.5 mM AMP, and 0.5 mM MgC1,. The product ribose 5-phosphate (ribose 5 -P ) was analyzed at the indicated times as described under "Experimental Procedures" and expressed as moles of product/mole of enzymatic catalytic sites. Ordinate values of 1 represent one catalytic turnover and divide the pre-steady-state and steady-state regions of catalysis.
nucleosidases from other sources (DeWolf et al., 1979;Leung and Schramm, 1980). Pre-steady State Kinetics of Native and A(l28-135)AMN-In contrast to the hysteresis of steady-state measurements (Fig. 8), rapid reaction kinetics at a catalytic site concentration of 10 pM resulted in a linear rate of product formation for the first turnover as well as two subsequent turnovers for both the native and A(128-135)AMN (Fig. 9). The reaction rate for native enzyme (Fig. 9A) gives a specific activity of 5.4 pmol/min/mg compared to a Vmax of 22 pmol/min/mg in steady-state measurements. The specific activity of the mutant enzyme was 0.09 pmol/min/mg for the first three turnovers compared to a V,,, of 2.1 pmol/min/mg in dilute solu-tion following the hysteretic conversion. With both the native and mutant enzymes, the reaction rates are slower than expected based on steady-state kinetics with dilute enzyme solutions. The ratio of activity for the mutant enzyme is 0.09/ 2.1 = 0.04 as a consequence of enzyme concentration while for native enzyme the ratio is 5.4/22 = 0.24. Thus, the response of catalytic efficiency to protein concentration differs significantly for the enzymes.

DISCUSSION
Peptide Analysis of Native and A(128-135)AMP Nucleosidase-A majority of the amino acid sequence information deduced from DNA sequencing for AMP nucleosidase has been confirmed by FABMS analysis of protease V8-trypsin digests of the protein. Of the 43 predicted peptides for native and mutant AMP nucleosidases in mass range 450-2,200, direct mass spectroscopic observation was achieved for 31 peptides. These included peptides distributed from amino acid 5 to the C terminus of the 483-amino acid chain. The sequences of peptides 1, 27, 28, 29, 52, and 53 were previously determined by N-terminal Edman sequencing as tryptic peptides. Thus, 37 of the 43 peptides within the observed mass range are confirmed. The peptides which were not detected by FABMS include the cysteine-containing peptides and peptides with masses above 2,200. Di-and most tripeptides were below mass 450 and were not included in the analysis. Cysteines were not protected in this protocol. No inconsistencies were found between the peptide mass analysis and the DNA sequence, except for a typographical error in the original report (Leung et al., 1989) which converts VaP5 to Gly75.
The structure of the mutated enzyme was confirmed from the DNA sequence and the direct observation of the new peptide VTlGm, which results from the elimination of Glu131, the site of a VB protease cleavage. Peptide VT16m replaces VT16 and VT17 of the normal enzyme. All predicted changes were readily observed in the FABMS data.
Properties of A(l28-135)AMN"The deduced amino acid sequences from yeast AMP deaminase and E. coli AMP nucleosidase were compared to test the hypothesis that the deaminase had evolved from the nucleosidase (Meyer et al., 1989). Both of these enzymes contain catalytic sites for AMP, allosteric activator sites for ATP, and allosteric inhibition by PO, which is competitive for the ATP sites. These enzymes provide the major pathway for AMP degradation in cell-free extracts of E. coli and S. cereuisiae. In the species tested, only prokaryotes contain AMP nucleosidase and only eukaryotes contain AMP deaminase (Merkler et al., 19891, suggesting an evolutionary replacement of AMP nucleosidase by AMP deaminase. Despite the circumstantial evidence for functional homology, only a single region of eight amino acids showed strong identity. Neither AMP nucleosidase nor AMP deaminase contain the consensus sequences for adenylate-binding sites. The amino acid sequences 128DGSELTLD in AMP nucleosidase and 344DGKLLTLD in AMP deaminase might be expected to play a functional role with respect to ATP binding, AMP binding, or catalysis or phosphate binding. Similar sequences are found in a family of proteins which contain binding sites for nucleotides, phosphate, or oligonucleotides (Table IV). In glyceraldehyde-3-phosphate dehydrogenase, this sequence forms a well-defined turn which is in contact with both the adenine and ribose rings of NAD+ at the catalytic site (Rossman et al., 1975). In the ras proteins, this sequence leads to the "phosphoryl group" region which makes contact with the magnesium chelated to the 7-phosphoryl of  Leung and Schramm (1980). described under "Experimental Procedures." purification procedures (Leung et al., 1989;Leung and Schramm, 1984).   Dayhoff, 1979) are indicated by a double dot under the amino acid. AMP nucleosidase is from E. coli, AMP deaminase from bakers' yeast, G-3-P dehydrogenase is from chloroplast, K-ras is human, RNA polymerase is from tomato spotted with virus, Frz E is from Myxococcus xonthus and is homologous to CheA and CheY of S. typhimurium and is also called gliding motility regulatory protein. PhoE is the phosphate-inducible outer membrane porin of E. coli, the K+-ATPase is from brine shrimp, LysA is from Bacillus subtilis, and nucleolin is from humans.  Leung et al., 1989Meyer et al., 1989Ferri et al., 1990Barbacid, 1987de Haan et al., 1991McCleary and Zusman, 1990 van der Ley and Tommassen, 1987Baxter-Lowe et al., 1989Yamamoto et al., 1991Srivastava et al., 1989 bound MgGTP (Barbacid, 1987;Schlichting et al., 1990). Asps7 in the sequence for K-ras in Table IV corresponds to the carboxylate which chelates the magnesium.

AMP
The relationship of amino acids 126-138 of AMP nucleosidase from E. coli to sequences known to interact with nucleotides, magnesium chelates, and phosphates in other proteins also suggested that this region may be a substrate or allosteric site. The allosteric activator is MgATP. AMP binds at the catalytic site and inorganic phosphate also binds in competition with MgATP. The inhibition constants for inorganic phosphate of 200 and 250 FM for native and A(128-135)AMP nucleosidases clearly indicated that no functional change had occurred at the phosphate-binding site as a result of this mutation. Likewise, the MgATP activation constants of 22 and 100 FM indicated no substantial difference in the ability of the mutant enzyme to be activated by MgATP. The saturation kinetics with AMP for both the mutant and native enzymes indicated that MgATP caused an apparent increase in AMP affinity by approximately two orders of magnitude.
Without ATP, the enzymes require approximately 20 mM AMP to reach half-V,,,.,. This constant drops to 90-210 g M when ATP is present. The substrate-binding site for AMP interaction is relatively unchanged by the mutation, with S0.s values differing only about 2-fold with or without activation by MgATP. Formycin 5-phosphate is a strong competitive inhibitor of AMP nucleosidases (DeWolf et al., 1979) and of the mutant enzyme. The binding of competitive inhibitors provides dissociation constants which are independent of the catalytic rate constants which influence K,,, and So.5 values for substrate. It is significant that there is little change in the Ki for formycin 5-phosphate, establishing that the mutation does not affect substrate analogue binding. Thus, none of the kinetic constants for substrates, activator, or inhibitor are strongly influenced by the deletion. The sequence so strongly implicated by homology search logic is therefore not required for binding of any known ligand to AMP nucleosidase.
In contrast to the kinetic constants relating to substrate, inhibitor, and effector binding, the catalytic efficiency of