Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon,Sulfolobus solfataricus: molecular cloning,nucleotide sequencing,expression in Escherichia coli,and characterization of the recombinant enzyme

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.     

Structure of the complex of yeast adenylate kinase with the inhibitor P1,P5-di(adenosine-5'-)pentaphosphate at 2.6 A resolution

Adenylate kinase from yeast cytosol was crystallized as a 1:1 complex with the inhibitor P1,P5-di(adenosine-5'-)pentaphosphate. The crystalline structure was solved by multiple isomorphous replacement at a resolution of 3 A (1 A = 0.1 nm) and subsequent structural refinement at 2.6 A resolution. The yeast enzyme belongs to the group of large variants among the adenylate kinases, whereas the structurally known porcine cytosolic enzyme is a small variant. A comparison showed that the additional 31-residue segment of the large variants covers the active center. This had not been expected, because small and large variants show similar enzyme kinetics. Apart from this insertion, the chain folds of both adenylate kinases are the same. The yeast enzyme with bound inhibitor, however, assumes a much more closed form. In relation to the porcine enzyme without substrate, a segment of 28 residues containing two helices is rotated by about 30 degrees, closing the deep cleft at the active center. This corresponds to the expected induced fit. Sequence comparisons with other adenylate kinases suggest that one of the adenosine moieties of the inhibitor does not bind at a native nucleotide-binding site of the enzyme.     

The switch between two conformations of adenylate kinase

Crystalline adenylate kinase from porcine muscle cytosol can assume two interconvertible structures. Here, we report the refined structure of crystal form B at 3.3 A resolution and compare it with crystal form A. Crystal forms A and B can be interconverted by protonation and deprotonation of His36, which is located deep in the active center cleft. The changes concern the molecular packing as well as the polypeptide chain conformation. On conversion from crystal form A to B, the N-terminal alpha-helix unwinds, the active center cleft opens to some extent and the nucleotide-binding glycine-rich loop 15-22 at the active center is detached from the bulk protein. This loop has counterparts in various important mononucleotide-binding proteins and is known to bind a phosphoryl group in adenylate kinase and in the oncogenic ras proteins. It is most likely involved in the phosphoryl transfer and the concomitant conformational changes. it is suggested that the two observed conformations are relevant for enzyme action in solution: they represent two of a series of three known snapshots depicting the enzyme during the substrate binding process.     

Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Evidence that the enzyme is a homodimer of identical subunits homologous to rat liver-specific alloantigen F.

4-Hydroxyphenylpyruvic acid dioxygenase is an important enzyme in tyrosine catabolism in most organisms. From porcine and human liver cDNA libraries we isolated complementary DNA inserts for the enzyme. Protein sequence analysis of the porcine enzyme revealed a block of the amino terminus of the mature enzyme. Comparison of the amino acid sequence determined by Edman degradation of peptides derived from porcine liver 4-hydroxyphenylpyruvic acid dioxygenase with the nucleotide sequences revealed the primary structure of the porcine and human enzymes. The mature human and porcine enzymes have an 89% amino acid sequence identity in amino acid residues and are composed of 392 amino acid residues. A computer-assisted homology search revealed that the enzyme is 88% identical in amino acid sequence to rat liver-specific alloantigen F. A monoclonal antibody (mob 51), which can immunoprecipitate both the human and porcine enzymes, was developed. Cultured BMT-10 cells transfected with the cDNA insert of the human enzyme, using the expression vector pCAGGSneodE, produced a polypeptide with an M(r) of 43,000, which was immunoprecipitated with mob 51. Enzymic activity of the enzyme was detected in the transfected cells but not in the mock transfected cells. These findings suggest that the human 4-hydroxyphenylpyruvic acid dioxygenase is a homodimer of two identical subunits with an M(r) of 43,000. Liver-specific alloantigen F seems to be closely related to the enzyme or possibly to the subunit of the enzyme itself. Elucidation of the complete amino acid sequence of the enzyme is expected to reveal structure-function relationships of this metabolically important enzyme and to shed light on inherited disorders related to tyrosine metabolism, especially tyrosinemia types 1 and 3.     

Mitochondrial adenylate kinase from chicken liver. Purification characterization and its cell-free synthesis

Mitochondrial adenylate kinase has been purified 5400-fold from chicken liver extract in an overall yield of 36%. The purified enzyme has a specific activity of 810 U/mg, a molecular weight of 28 000, and the following amino acid composition: 21 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 16 proline, 22 glycine, 22 alanine, 15 valine, 6 methionine, 11 isoleucine, 29 leucine, 5 tyrosine, 7 phenylalanine, 16 lysine, 7 histidine, 19 arginine, 3 half-cystine, and no tryptophan, totalling 257 residues. The purified enzyme has one disulfide bond and one sulfhydryl group. The disulfide bond is related to the active conformation of the enzyme, whereas the sulfhydryl group does not contribute to the enzyme activity. The sulfhydryl group is easily oxidized in the presence of Cu2+ resulting in the formation of dimer with about one half of the specific activity of the monomer. The enzyme is similar to porcine heart mitochondrial adenylate kinase in antigenicity but different from chicken cytosolic adenylate kinase. Mitochondrial adenylate kinase was synthesized in the mRNA-dependent rabbit reticulocyte lysate system programmed with total chicken liver RNA. The mobility in sodium dodecylsulfate gel electrophoresis of the product obtained in vitro was the same as that of the purified mitochondrial adenylate kinase. This evidence indicates that the mitochondrial adenylate kinase is synthesized as a polypeptide with a molecular weight indistinguishable from that of the mature protein.     

Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver

Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.     

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei

In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE. The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable. The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.     

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.     

Studies on tyrosine residues in porcine muscle adenylate kinase. Circular dichroism spectra and chemical modification with tetranitromethane.

Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.     

Refined structure of porcine cytosolic adenylate kinase at 2.1 A resolution

The crystal structure of porcine cytosolic adenylate kinase has been established at 2.1 A resolution using a restrained least-squares refinement method. Based on 11,251 independent reflections of better than 10 A resolution, a final R-factor of 19.3% was obtained with a model obeying standard geometry within 0.026 A in bond lengths and 3.3 degrees in bond angles. In comparison with the previous structure at 3 A resolution, there is a significant improvement. The high resolution structure has been used to rationalize the strictly conserved residues in the adenylate kinase family. Among these is the glycine-rich loop, which forms a giant anion hole accommodating a sulfate ion which mimics a phosphoryl group of a substrate. Such a structure seems to occur in a large group of mononucleotide binding proteins. Moreover, a conserved cis-proline has been detected in the active center. A structural comparison with the complex between adenylate kinase from yeast and a substrate-analog at medium resolution indicates that this kinase performs appreciable mechanical movements during a catalytic cycle. The reported structure presumably represents an open form of the enzyme, similar to that in solution in the absence of substrates. However, since there are large intermolecular contacts in the crystal, some deviation from the solution structure has to be expected.     

The structural isomerisation of human-muscle adenylate kinase as studied by 1H-nuclear magnetic resonance

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H-nuclear magnetic resonance spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947-6954]. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37-45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5')pentaphospho(5')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion-controlled process kon being 10(8) M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.     

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

The sialate-pyruvate lyase from pig kidney. Elucidation of the primary structure and expression of recombinant enzyme activity.

The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.     

Partial purification and properties of the two common inherited forms of human erythrocyte adenylate kinase

1. The partial purification of adenylate kinase, types 1 and 2, from human erythrocytes is described. 2. Gel chromatography of both forms of the enzyme gave estimates of the molecular weights in the range 20000-23000. 3. Studies on crude haemolysates at various pH values indicated that the type 2 enzyme was less stable than the type 1. Heat denaturation studies on the partially purified enzymes confirmed these findings. 4. Measurements of rates of inhibition by iodoacetate and iodoacetamide showed that the type 2 enzyme reacts more readily than the type 1 enzyme with both reagents. 5. The effect of temperature on the initial velocity of ADP formation was measured at a single concentration of both AMP and MgATP(2-). The two forms of the enzyme responded differently to increasing temperature.     

Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2-nitro-5-thiocyanobenzoic acid: analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues

Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively.     

Circular dichroism investigation of Escherichia coli adenylate kinase

We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase. The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Barzu, O. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 5798-5802). We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214). The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R. H. (1974) Nature 250, 120-123). Carboxymethylation of wild type protein did not greatly affect the CD spectrum. The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%). Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C. Isolated C1 and C2 peptides had a large proportion of unordered structures. When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase. The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity.