Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.     

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.     

Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.

To investigate the ability of a protein to accommodate potentially destabilizing amino acid substitutions, and also to investigate the steric requirements for catalysis, proline was substituted at different sites within the long alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4 lysozyme. Of the four substitutions attempted, three yielded folded, functional proteins. The catalytic activities of these three mutant proteins (Q69P, D72P, and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12 degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals isomorphous with wild-type allowing the structure to be determined at high resolution. In the crystal structure of wild-type lysozyme the interdomain alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure the introduction of the proline causes this bend angle to increase to 14 degrees and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal domain relative to the amino-terminal one. Except for the immediate location of the proline substitution there is very little change in the geometry of the interdomain alpha-helix. The results support the view that protein structures are adaptable and can compensate for potentially destabilizing amino acid substitutions. The results also suggest that the precise shape of the active site cleft of T4 lysozyme is not critical for catalysis.     

Effect of foreign N-terminal residues on the conformational stability of human lysozyme.

To minutely understand the effect of foreign N-terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N-terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N-terminal Lys residue (Met(-1), Gly(-1) and Pro(-1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild-type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (DeltaDeltaG) were -9.1 to -12.2 kJ.mol-1. However, the stability of two single substitutions at the N-terminal slightly decreased; the DeltaDeltaG values were only -0.5 to -2.5 kJ.mol-1. The results indicate that human lysozyme is destabilized by an expanded N-terminal residue. The crystal structural analyses of K1M, K1A and Gly(-1) revealed that the introduction of a residue at the N-terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.     

Role of proline residues in human lysozyme stability: a scanning calorimetric study combined with X-ray structure analysis of proline mutants.

It has been shown that protein stability can be modulated from site-directed mutations that affect the entropy of protein unfolding [Matthews, B. W., Nicholson, H., & Becktel, W. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6663-6667]. However, the effect of a specific amino acid replacement on stability highly depends on the location of the mutation site and its environment in the protein structure [Yutani, K., Hayashi, S., Sugisaki, Y., & Ogasahara, K. (1991) Proteins Struct., Funct., Genet. 9, 90-98). To clarify the role of specific proline residues in the thermostability of human lysozyme (h-lysozyme), a series of proline mutants were investigated by means of scanning calorimetry and high-resolution X-ray crystallography. The thermodynamic properties of the mutant and wild-type h-lysozymes are compared and discussed on the basis of their three-dimensional structure. h-Lysozyme contains two proline residues at positions 71 and 103. The Pro71----Gly substitution was found to destabilize h-lysozyme by decreasing the entropic contribution of unfolding by about 2 kcal/mol at 68.8 degrees C. This is consistent with the theoretical expectations for such a substitution. However, the same substitution at position 103 (Pro103----Gly) does not affect h-lysozyme stability, and the thermodynamic properties of the P71G/P103G and P71G mutants are essentially the same. Pro71 which is conserved among lysozymes from other species, appears to be important for stability, whereas Pro103, which is not conserved, does not. These differences are explained in terms of residue accessibility to the solvent and crystallographic B-factor, which reflects the amino acid mobility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gly or Ala substitutions for Pro(210)Thr(211)Asn(212) at the β8-β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

A comparison of the primary structures among psychrophilic, mesophilic, and thermophilic subtilases revealed that the turn between the β8 and β9 strands (β8-β9 turn, BPN' numbering) of psychrophilic subtilases are more flexible than those of their mesophilic and thermophilic counterparts. To investigate the relationship between structure of this turn and enzyme activity as well as thermostability of mesophilic subtilisin Carlsberg (sC), we analyzed 6 mutants of sC with a single, double, or triple Gly or Ala substitutions for Pro(210)Thr(211)Asn(212) at the β8-β9 turn. Among the single Gly substitutions, the P210G substitution most significantly (1.5-fold) increased the specific activity on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) substrate and 12-fold decreased the thermostability. All mutants tested showed the increased k(cat) for the AAPF substrate and reduced thermostability compared with the wild-type sC. The k(cat) values of the P210G, P210G/T211G, and P210G/T211G/N212G mutants were 1.5-, 1.7-, and 1.8-fold higher than that of the wild-type sC. There were significant positive correlations between k(cat) and thermal inactivation rates as well as k(cat) and K(m) of the wild-type and mutants. These results demonstrate that the structure of β8-β9 turn, despite its distance from the active site, has significant effects on the catalytic rate and thermostability of sC through a global network of intramolecular interactions and suggest that the lack of flexibility of this turn stabilizes the wild-type sC against thermal inactivation in compensation for some loss of catalytic activity.     

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.     

Stabilization of E. coli Ribonuclease HI by the 'stability profile of mutant protein' (SPMP)-inspired random and non-random mutagenesis

The change in the structural stability of Escherichia coli ribonuclease HI (RNase HI) due to single amino acid substitutions has been estimated computationally by the stability profile of mutant protein (SPMP) [Ota, M., Kanaya, S. Nishikawa, K., 1995. Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H. J. Mol. Biol. 248, 733-738]. As well, an effective strategy using random mutagenesis and genetic selection has been developed to obtain E. coli RNase HI mutants with enhanced thermostability [Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., Kanaya, S., 1994. A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol. Chem. 269, 26904-26911]. In this study, both methods were combined: random mutations were individually introduced to Lys99-Val101 on the N-terminus of the alpha-helix IV and the preceding beta-turn, where substitutions of other amino acid residues were expected to significantly increase the stability from SPMP, and then followed by genetic selection. Val101 to Ala, Gln, and Arg mutations were selected by genetic selection. The Val101-->Ala mutation increased the thermal stability of E. coli RNase HI by 2.0 degrees C in Tm at pH 5.5, whereas the Val101-->Gln and Val101-->Arg mutations decreased the thermostability. Separately, the Lys99-->Pro and Asn100-->Gly mutations were also introduced directly. The Lys99-->Pro mutation increased the thermostability of E. coli RNase HI by 1.8 degrees C in Tm at pH 5.5, whereas the Asn100-->Gly mutation decreased the thermostability by 17 degrees C. In addition, the Lys99-->Pro mutation altered the dependence of the enzymatic activity on divalent metal ions.     

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme.     

Exploring Nonnatural Evolutionary Pathways by Saturation Mutagenesis: Rapid Improvement of Protein Function

Random point mutagenesis does not access a large fraction of protein sequence space corresponding to primarily nonconservative amino acid substitutions. The cost of this limitation during directed evolution is unknown. Random point mutagenesis over the entire gene encoding the psychrophilic protease subtilisin S41 identified a pair of residues (Lys211 and Arg212) where mutations provided significant increases in thermostability. These were subjected to saturation mutagenesis to test whether the amino acids not easily accessible by point mutagenesis provide even better ``solutions' to the thermostabilization challenge. A significant fraction of these variants surpassed the stability of the variants with point mutations. DNA sequencing revealed highly hydrophobic residues in the four most stable variants (Pro/Ala, Pro/Val, Leu/Val, and Trp/Ser). These nonconservative replacements, accessible only by multiple (two to three) base substitutions in a single codon, would be extremely rare in a point mutation library. Such replacements are also extremely rare in natural evolution. Saturation mutagenesis may be used advantageously during directed evolution to explore nonnatural evolution pathways and enable rapid improvement in protein traits. Received: 15 March 1999 / Accepted: 28 June 1999     

Comparison of repetitive sequences derived from high molecular weight subunits of wheat glutenin, an elastomeric plant protein

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.     

Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase

Loop 52-72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k(cat) by up to 3-fold, with no effect on the K(m) for fructose 1,6-bisphosphate. The K(a) for Mg(2+) in the Lys(50) --> Pro mutant increases approximately 15-fold, whereas that for the Ala(51) --> Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2'(3')-O-(trinitrophenyl)adenosine 5'-monophosphate, the K(i) for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys(50) --> Pro changes the mechanism of AMP inhibition with respect to Mg(2+) from competitive to noncompetitive and abolishes K(+) activation. The K(i) for fructose 2,6-bisphosphate increases approximately 20- and 30-fold in the Lys(50) --> Pro and Ala(51) --> Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr(57) suggests an altered conformational state for Loop 52-72 due to the proline at position 50. Evidently, the Pro(50) mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled.     

Protein thermostabilization by proline substitutions

Many recent approaches involving site-directed mutants have succeeded in increasing the thermostability of proteins. It is well known that replacements with proline residues reduce the conformational degrees of freedom in the main polypeptide chain and thus can increase protein thermostabilization. We have studied protein thermostabilization by introducing proline substitutions in the homologous oligo-1,6-glucosidases from various Bacillus strains which grow within different temperature ranges. As a consequence, the `proline rule' was proposed for protein thermostabilization. The principle of this rule is that an increase in the frequency of proline occurrence at β-turns and/or an increase in the total number of hydrophobic residues can enhance protein thermostability. We have generated several lines of evidence supporting the theory from the comparative analysis of oligo-1,6-glucosidases in their primary and secondary structures and molecular properties, the X-ray crystal structure analysis of the Bacillus cereus oligo-1,6-glucosidase, and the enhancement in thermostability of the oligo-1,6-glucosidase by cumulative replacements with prolines. As a new finding from the studies, two specific sites (second positions at β-turns and N1 positions of α-helices) were found to be the most critical to protein thermostabilization dependent on several structural prerequisites for proline substitution.     

Role of conserved proline residues in stabilizing tryptophan synthase alpha subunit: analysis by mutants with alanine or glycine

To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.     

Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease.

Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis. One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop. The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements. Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates. When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease. Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species. Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein. These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins. The possible mechanisms of protein stabilization are also discussed.     

Evidence regarding the hypothesis that the histidine-histidine contact pairs may affect protein stability

It has been lately proposed that the interaction between like-charged residues stabilizes the native state of proteins. To explore this, we created a histidine-histidine pair in the Ca-III binding site of the Bacillus amyloliquefaciens α-amylase (BAA) and then examined the impact of this pairing on the BAA. For this purpose, we used site-directed mutagenesis (SDM) to substitute Pro407 with His, Ala, Gln, Arg, and Glu in the BAA. Subsequently, thermostability, kinetic parameters and structural properties of these variants were measured. Moreover, His-His pairing effect on the BAA thermostability was examined by simultaneous mutation of two residues (P407H/H406A and P407H/H406N). The data exhibited a significant improve in thermostability and structural features of enzyme by His replacement instead of Pro407. Other substitutions in this site did not have a significant effect on the enzyme properties, except for P407R, which yielded a partial improvement. The results also showed that the thermostabilities of double mutants significantly decreased compared with that of the P407H mutant. Moreover, the thermostability of P407H remarkably increased compared with that of other variants even in the absence of Ca(2+). Our data clearly demonstrated that His406-His407 pairing was the major cause for improved thermal stability.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis.

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.