Single proline substitutions in predicted alpha-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation

Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.     

Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.     

Protein structural influences in rhodopsin evolution

Incorporating specific structural information can be important for developing a realistic model of evolution for phylogenetic reconstruction of protein-coding genes. We analyzed 62 sequences of vertebrate rhodopsin. The bovine rhodopsin structure was used to label residue sites by surface accessibility, secondary structure, and transmembrane (TM) location. Residue sites with amino acid differences were identified; using maximum parsimony (MP), homoplasious residues were identified. Residues were analyzed for patterns that would indicate correlation of rate with secondary structure, surface accessibility, or position relative to the lipid bilayer. Surface residues, especially those residing in one of the seven TM helices, were significantly correlated with high rates of amino acid substitution. This category of residues, defined solely by protein structural characteristics, potentially defined a class enriched in homoplasious residues. MP analysis using all sites led to a tree with anomalies in the relationships of amphibian, mammalian, bird, and alligator species. Analysis excluding the structurally defined residue class recovered a more accurate phylogeny. A model is presented for including structural influences on rate in phylogenetic inference.     

Positive charge intrinsic to Arg(37)-Arg(38) is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3, 4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg(37)-Arg(38) of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg(37)-Arg(38) site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N-terminal residues 1-60 by computer software specified the location of the Arg(37)-Arg(38) sequence in the turn intervening between the two alpha-helices (residues 16-29 and residues 41-59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究

非解朊栖热菌HG10 2耐热 β-糖苷酶为 (β/α)₈桶状结构 ,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较 ,推测Glu164和Glu338分别是质子供体和亲核基团两个活性位点 ;在α-螺旋N端第一位的脯氨酸和蛋白质外周的精氨酸是耐热机制的关键位点和关键氨基酸残基。为确定这些氨基酸残基的功能 ,通过基因定点突变的方法分别把Glu164、Glu338、Pro316、Pro356、Pro344和Arg325置换成Gln、Ala、Gly、Ala、Phe和Leu ,同时还对Pro316和Pro356进行了双置换。突变酶经过纯化得到电泳纯 ,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析 ,结果表明Glu164和Glu338分别是质子供体和亲核基团 ,亲核基团的突变酶TnglyE338A可以合成混合型糖苷键寡糖类似物 ;在α-螺旋N端第一位的Pro316和Pro356以及在蛋白质外周形成离子键的Arg325均是对耐热性有贡献的关键氨基酸残基。     

Exploring the environmental preference of weak interactions in (alpha/beta)8 barrel proteins

The environmental preference for the occurrence of noncanonical hydrogen bonding and cation-pi interactions, in a data set containing 71 nonredundant (alpha/beta)(8) barrel proteins, with respect to amino acid type, secondary structure, solvent accessibility, and stabilizing residues has been performed. Our analysis reveals some important findings, which include (a) higher contribution of weak interactions mediated by main-chain atoms irrespective of the amino acids involved; (b) domination of the aromatic amino acids among interactions involving side-chain atoms; (c) involvement of strands as the principal secondary structural unit, accommodating cross strand ion pair interaction and clustering of aromatic amino acid residues; (d) significant contribution to weak interactions occur in the solvent exposed areas of the protein; (e) majority of the interactions involve long-range contacts; (f) the preference of Arg is higher than Lys to form cation-pi interaction; and (g) probability of theoretically predicted stabilizing amino acid residues involved in weak interaction is higher for polar amino acids such as Trp, Glu, and Gln. On the whole, the present study reveals that the weak interactions contribute to the global stability of (alpha/beta)(8) TIM-barrel proteins in an environment-specific manner, which can possibly be exploited for protein engineering applications.     

Elucidating the protein cold-adaptation: Investigation of the parameters enhancing protein psychrophilicity

To investigate the role of the critical parameters in adaptation of proteins to low temperatures, a comparative systematic analysis was performed. Several parameters were proposed to have contribution to cold adaptation of proteins. Among proposed parameters, total values of residual structure states, secondary structure states and oligomeric states were alike in both psychrophilic and mesophilic proteins. In addition, our results provided new quantitative information about the trends in the substitution preference of Ile, Phe, Tyr, Lys, Arg, His, Glu and Leu with most of amino acids and substitution avoidance of Gly, Thr and Ala with most of amino acids. These findings would help future efforts propose a strategy for designing psychrophilic proteins.     

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.     

Discarding functional residues from the substitution table improves predictions of active sites within three-dimensional structures

Substitutions of individual amino acids in proteins may be under very different evolutionary restraints depending on their structural and functional roles. The Environment Specific Substitution Table (ESST) describes the pattern of substitutions in terms of amino acid location within elements of secondary structure, solvent accessibility, and the existence of hydrogen bonds between side chains and neighbouring amino acid residues. Clearly amino acids that have very different local environments in their functional state compared to those in the protein analysed will give rise to inconsistencies in the calculation of amino acid substitution tables. Here, we describe how the calculation of ESSTs can be improved by discarding the functional residues from the calculation of substitution tables. Four categories of functions are examined in this study: protein–protein interactions, protein–nucleic acid interactions, protein–ligand interactions, and catalytic activity of enzymes. Their contributions to residue conservation are measured and investigated. We test our new ESSTs using the program CRESCENDO, designed to predict functional residues by exploiting knowledge of amino acid substitutions, and compare the benchmark results with proteins whose functions have been defined experimentally. The new methodology increases the Z-score by 98% at the active site residues and finds 16% more active sites compared with the old ESST. We also find that discarding amino acids responsible for protein–protein interactions helps in the prediction of those residues although they are not as conserved as the residues of active sites. Our methodology can make the substitution tables better reflect and describe the substitution patterns of amino acids that are under structural restraints only.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase. Localized replacements in the carboxyl-terminal alpha-helix cause marked alterations in allosteric properties and intersubunit interactions.

A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax. In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km. Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.     

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Beta-breakers: an aperiodic secondary structure

We have studied the architecture of parallel beta-sheets in proteins and focused on the residues that initiate and terminate the beta-strands. These beta-breaker residues are at the origin of the kink between the beta-strand and the turn that precedes or follows it. beta-Breakers can be located automatically using a consensus approach based on algorithmic secondary structure assignment, solvent accessibility and backbone dihedral angles. These beta-breakers are conformationally homogeneous with respect to side-chain solvent accessibility and backbone dihedral angle profile. A sequence-structure correlation is noted: a restricted subset of amino acids is observed at these positions. Analysis of homologous protein sequences shows that these residues are more highly conserved than other residues in the loop. We conclude that beta-breakers are the structural analogs of the N and C-terminal caps of alpha-helices. The identification of this aperiodic substructure suggests a strategy for improving secondary structure prediction and may guide site-directed mutagenesis experiments.     

Refined crystal structure of dogfish M4 apo-lactate dehydrogenase

The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.     

A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.     

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.