Biomedical subjects

Hideyuki Hayashi

Publications and source records attributed to Hideyuki Hayashi.

At least 55 records · Page 3Linked to original sources

PubMed2003

Crystal structures of threonine synthase from Thermus thermophilus HB8: conformational change, substrate recognition, and mechanism.

Threonine synthase, which is a PLP-dependent enzyme, catalyzes the beta,gamma-replacement reaction of l-homoserine phosphate to yield threonine and inorganic phosphate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its unliganded form and complexed with the substrate analogue 2-amino-5-phosphonopentanoic acid have been determined at 2.15 and 2.0 A resolution, respectively. The complexed form, assigned as an enamine, uncovered the interactions of the cofactor-analogue conjugate with the active site residues. The binding of the substrate analogue induces a large conformational change at the domain level. The small domain rotates by about 25 degrees and approaches the large domain to close the active site. The complicated catalytic process of the enzyme has been elucidated based on the complex structure to reveal the stereochemistry of the reaction and to present the released inorganic phosphate as a possible catalyst to carry a proton to the Cgamma atom of the substrate.

Binding Sites↗

PubMed2003

Strain and catalysis in aspartate aminotransferase.

The notion of "ground-state destabilization" has been well documented in enzymology. It is the unfavourable interaction (strain) in the enzyme-substrate complex, and increases the k(cat) value without changing the k(cat)/K(m) value. During the course of the investigation on the reaction mechanism of aspartate aminotransferase (AAT), we found another type of strain that is crucial for catalysis: the strain of the distorted internal aldimine in the unliganded enzyme. This strain raises the energy level of the starting state (E+S), thereby reducing the energy gap between E+S and ES(++) and increasing the k(cat)/K(m) value. Further analysis on the reaction intermediates showed that the Michaelis complex of AAT with aspartate contains strain energy due to an unfavourable interaction between the main chain carbonyl oxygen and the Tyr225-aldimine hydrogen-bonding network. This belongs to the classical type of strain. In each case, the strain is reflected in the pK(a) value of the internal aldimine. In the historical explanation of the reaction mechanism of AAT, the shifts in the aldimine pK(a) have been considered to be the driving forces for the proton transfer during catalysis. However, the above findings indicate that the true driving forces are the strain energy inherent to the respective intermediates. We describe here how these strain energies are generated and are used for catalysis, and show that variations in the aldimine pK(a) during catalysis are no more than phenomenological results of adjusting the energy levels of the reaction intermediates for efficient catalysis.

Aspartate Aminotransferases↗

PubMed2003

Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme.

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity, and purified SPT to homogeneity. Unlike eukaryotic enzymes, this enzyme was a water-soluble homodimeric protein. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT showed about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The purified recombinant-SPT showed the characteristic absorption spectrum derived from its coenzyme pyridoxal 5'-phosphate (PLP). The addition of the substrate, L-serine, caused spectral changes indicating the formation of the external aldimine intermediate. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.

Acyltransferases↗

PubMed2003

Characterization of histidinol phosphate aminotransferase from Escherichia coli.

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.

Escherichia coli↗

PubMed2003

Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme.

Branched-chain amino acid aminotransferase (BCAT), which has pyridoxal 5'-phosphate as a cofactor, is a key enzyme in the biosynthetic pathway of hydrophobic amino acids (leucine, isoleucine, and valine). The enzyme reversibly catalyzes the transfer of the amino group of a hydrophobic amino acid to 2-oxoglutarate to form a 2-oxo acid and glutamate. Therefore, the active site of BCAT should have a mechanism to enable recognition of an acidic amino acid as well as a hydrophobic amino acid (double substrate recognition). The three-dimensional structures of Escherichia coli BCAT (eBCAT) in complex with the acidic substrate (glutamate) and the acidic substrate analogue (glutarate) have been determined by X-ray diffraction at 1.82 and 2.15 A resolution, respectively. The enzyme is a homo hexamer, with the polypeptide chain of the subunit folded into small and large domains, and an interdomain loop. The eBCAT in complex with the natural substrate, glutamate, was assigned as a ketimine as the most probable form based upon absorption spectra of the crystal complex and the shape of the residual electron density corresponding to the cofactor-glutamate bond structure. Upon binding of an acidic substrate, the interdomain loop approaches the substrate to shield it from the solvent region, as observed in the complex with a hydrophobic substrate. Both the acidic and the hydrophobic side chains of the substrates are bound to almost the same position in the pocket of the enzyme and are identical in structure. The inner side of the pocket is mostly hydrophobic to accommodate the hydrophobic side chain but has four sites to coordinate with the gamma-carboxylate of glutamate. The mechanism for the double substrate recognition observed in eBCAT is in contrast to those in aromatic amino acid and histidinol-phosphate aminotransferases. In an aromatic amino acid aminotransferase, the acidic side chain is located at the same position as that for the aromatic side chain because of large-scale rearrangements of the hydrogen bond network. In the histidinol-phosphate aminotransferase, the acidic and basic side chains are located at different sites and interact with different residues of the disordered loop.

Binding Sites↗

PubMed2003

Effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses in mice.

The present study was undertaken to study the effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses. For this study, mice were immunized by s.c. injection of ovalbumin (OVA) emulsified with complete Freund's adjuvant into the base of the tail (day 0). Varying doses of indomethacin were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-gamma as an indicator of Th1 responses and anti-OVA IgG1 and interleukin-10 as that of Th2 responses were measured. The results showed that treatment with indomethacin was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Indomethacin inhibited both Th1 and Th2 responses, although the nonsteroidal anti-inflammatory drug suppressed the former more effectively than the latter. Administration of indomethacin resulted in suppression of antigen (OVA)-induced arthritis that was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that nonsteroidal anti-inflammatory drugs may downregulate Th1 and, to a lesser extent, Th2 immune responses.

Animals↗

PubMed2003

Aging changes in apparent accommodation in eyes with a monofocal intraocular lens.

PURPOSE: To examine the aging changes in the amplitude of apparent accommodation in eyes with a monofocal intraocular lens (IOL). DESIGN: Prospective comparative observational study. METHODS: Two hundred eyes of 200 patients who were scheduled for phacoemulsification and monofocal IOL implantation were studied. Forty patients in each of five age groups-younger than 40 years of age or in their 50s, 60s, 70s, and 80s-were prospectively recruited. Using an accommodopolyrecorder, the amplitude of apparent accommodation of these patients was measured approximately 1 month after surgery. Visual acuities from far to near distances after best-corrected distance were examined using an all-distance vision tester, and the region of accommodation at which each patient achieved a visual acuity of 20/29 or 20/40 was determined and converted to the diopteric range. Contrast sensitivity was also examined using a vision contrast test system. RESULTS: The amount of apparent accommodation as measured with the accommodopolyrecorder was decreased significantly in proportion to patient age (P <.0001). Furthermore, significant negative correlation was found between the amount of apparent accommodation and actual age of each patient (r = -.491). The diopteric range of accommodation at which the patients achieved 20/29 or 20/40 also reduced in proportion to the age. Although no significant differences were found in the mean far visual acuities between age groups, the intermediate and near visual acuities worsened significantly with age. The contrast sensitivity was also reduced in proportion to the age. CONCLUSIONS: The amplitude of apparent accommodation in eyes with a monofocal IOL decreases significantly in proportion to age, resulting in worsening of best-corrected intermediate and near visual acuities.

Accommodation, Ocular↗

PubMed2003

Correlation between posterior capsule opacification and visual function before and after Neodymium: YAG laser posterior capsulotomy.

PURPOSE: To investigate the correlation between visual acuity, contrast sensitivity, and contrast sensitivity with glare source (glare sensitivity), and the degree of posterior capsule opacification (PCO) before and after neodymium:yttrium-aluminum-garnet (Nd:YAG) laser posterior capsulotomy. DESIGN: Prospective interventional case series. METHODS: Eighty-four patients (90 pseudophakic eyes) scheduled to undergo Nd:YAG laser capsulotomy were recruited. Visual acuity and contrast sensitivity with and without the presence of a circular glare source (using the contrast glare tester) were measured before and after Nd:YAG laser capsulotomy. The PCO density value was measured using a Scheimpflug videophotography system. The visual functions were statistically correlated with the PCO value. RESULTS: Before Nd:YAG laser capsulotomy, strong correlation existed between the PCO value and visual acuity (r = 0.728). Contrast sensitivity and glare sensitivity were also weakly correlated with the PCO value, but the correlation coefficients were smaller than that of visual acuity. After Nd:YAG laser capsulotomy, mean visual acuity, contrast sensitivity, and glare sensitivity at all visual angles improved significantly (P <.0001). After capsulotomy, no significant correlation was found between visual acuity, contrast sensitivity, or glare sensitivity and the PCO value. CONCLUSIONS: Before Nd:YAG laser capsulotomy, visual functions correlated significantly with the degree of PCO. Specifically, visual acuity has a stronger association with PCO than does contrast sensitivity or glare sensitivity and therefore should be considered to reflect most accurately the degree of PCO. After capsulotomy, these visual functions improve markedly and no longer have a significant correlation with PCO.

Aged↗

PubMed2003

Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36.

The induction of fibroblast apoptosis and their clearance by phagocytes is essential for normal wound healing and prevention of scarring. However, little is known about the clearance of apoptotic fibroblasts and whether apoptotic cells are active participants in the recruitment and activation of phagocytes. In this study, we provide the first evidence that apoptotic fibroblasts actively release increased amounts of thrombospondin (TSP1) to actively recruit macrophages. Expression of TSP1 and its receptor CD36 was increased on the surface of apoptotic fibroblasts. By chemical cross-linking and immunoprecipitation we show that TSP1 and CD36 were directly associated. This was confirmed by confocal microscopy. Blockade of either CD36 or TSP1 on apoptotic fibroblasts inhibited phagocytosis. Blockade of alpha v beta 3 integrins as well as CD36 and TSP1 on macrophages inhibited phagocytosis. In contrast, phosphatidylserine or lectins were not involved. These findings suggest that apoptotic fibroblasts release TSP1 as a signal to recruit macrophages while the up-regulated expression of the CD36/TSP1 complex on their cell surface may form a ligand bridging the fibroblast to a complex consisting of alpha v beta 3/CD36/TSP1 on macrophages. These results establish fundamental mechanisms for the clearance of apoptotic fibroblasts and may provide insights into the processes involved in normal wound repair.

Antigens, CD↗

PubMed2003

Outcomes of surgery for posterior polar cataract.

PURPOSE: To report the preferred technique and the outcomes of surgery for posterior polar cataract. SETTING: Hayashi Eye Hospital, Fukuoka, Japan. METHODS: Medical records of 28 eyes of 20 consecutive patients with posterior polar cataract who had cataract surgery were reviewed. The surgical techniques used, intraoperative complications, preoperative and postoperative visual acuities, and the causes of impaired visual acuity after surgery were examined. RESULTS: Of the 28 eyes, 25 (89.3%) with a small to medium posterior polar opacity had standard phacoemulsification or aspiration surgery. Two eyes (7.1%) with a large opacity and soft lens nucleus had pars plana lensectomy, and 1 eye (3.6%) with a large opacity and hard nucleus had intracapsular cataract extraction. Among the eyes having phacoemulsification or aspiration surgery, posterior capsule rupture occurred in 2 (7.1%). The mean visual acuity improved significantly after surgery (P <.0001); however, the postoperative visual acuity was worse than 20/20 in 7 eyes (25.0%). The cause of the low acuity was amblyopia in 4 eyes (14.3%), impaired foveal function after retinal detachment in 2 eyes (7.1%), and macular degeneration in 1 eye (3.6%). CONCLUSION: Posterior polar cataracts can be safely extracted if the appropriate surgical technique is selected. Visual acuity improved significantly in most cases, although some patients with a unilateral cataract had previously developed amblyopia.

Adult↗

PubMed2003

Effects of bisphenol A on antigen-specific antibody production, proliferative responses of lymphoid cells, and TH1 and TH2 immune responses in mice.

1. We investigated the effect of bisphenol A (BPA), which binds estrogen receptors, on immune responses including production of antigen-specific antibodies, proliferative responses of lymphoid cells, and Th1 and Th2 responses. 2. For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 micro g kg(-1) of BPA immediately after immunization with hen egg lysozyme (HEL) (day 0) and then daily by day 20. On day 21, anti-HEL IgG antibodies in sera and proliferative responses of spleen cells to the antigen were measured. Anti-HEL IgG2a antibodies and IFN-gamma secreted from splenic lymphocytes were also measured as indicators of Th1 immune responses, while anti-HEL IgG1 antibodies and IL-4, as those of Th2 responses. 3. The results showed that treatment with 3000 micro g kg(-1) of BPA was followed by a significant increase in anti-HEL IgG as well as the antigen-specific cell proliferation. Anti-HEL IgG2a production and IFN-gamma secretion were significantly enhanced in mice treated with 300 and 30 micro g kg(-1) of BPA, respectively, while anti-HEL IgG1 production and IL-4 secretion were augmented in animals given 3000 and 300 micro g kg(-1) of the chemical, respectively. 4. Augmentation of these immune responses was also observed in mice exposed to 0.3-30 micro g kg(-1) of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses. 5. These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

Animals↗

PubMed2003

Increased soluble interleukin-6 receptor in vitreous fluid of proliferative vitreoretinopathy.

PURPOSE: To clarify the effect of the interaction between interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on retinal pigment epithelial (RPE) cell proliferation in proliferative vitreoretinopathy (PVR). METHODS: Concentrations of IL-6 and sIL-6R molecules in vitreous fluids were measured in eyes with PVR and idiopathic macular hole (MH), and the localization of IL-6 and IL-6R on the PVR fibrous membrane was studied. Production of IL-6 and sIL-6R by cultured RPE cells and the effect of the IL-6/sIL-6R complex on growth of cultured RPE cells were analyzed. RESULTS: Positive staining of IL-6 and IL-6R was observed in proliferating membranes in all PVR cases. IL-6 and sIL-6R concentrations in vitreous fluid from eyes with PVR were significantly higher than in eyes with MH (p < 0.05). A time-dependent increase in IL-6 molecules was identified in the culture medium of RPE cells, although sIL-6R was not detected. Dose-dependent growth of RPE cells was observed in the three concentrations (50, 100, and 500 ng/ml) of sIL-6R used. CONCLUSION: IL-6 derived from blood during the breakdown of the blood-retinal barrier (BRB) and produced by RPE cells and hematogenous sIL-6R cause RPE proliferation.

Cell Division↗

PubMed2003

Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids.

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH(+) and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).S. The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001-1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the alpha-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).SH(+). It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.

Aspartate Aminotransferases↗

PubMed2003

Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.

A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.

Amino Acid Sequence↗

PubMed2003

Chronic aortic dissection complicated by disseminated intravascular coagulation: successful treatment with endovascular stent-grafting.

PURPOSE: To report endovascular repair of a chronic aortic dissection complicated by disseminated intravascular coagulation (DIC). CASE REPORT: A 61-year-old man developed DIC associated with a chronic Stanford type B aortic dissection that occurred during cardiac catheterization 12 years earlier. At the current admission, computed tomography showed a partially thrombosed false lumen extending from the aortic arch to the left common iliac artery. On angiography, entry and re-entry tears were identified at the right subclavian and left common iliac arteries, respectively. After stent-graft implantation at the entry and re-entry sites, not only was the false lumen completely thrombosed but the DIC also resolved. The patient is doing well with no complication at 16 months after treatment. CONCLUSIONS: Endovascular stent-grafting is an acceptable alternative to surgical repair for aortic dissection accompanied by DIC.

Aortic Dissection↗

PubMed2003

Effect of methotrexate on Th1 and Th2 immune responses in mice.

We investigated the effect of the anti-rheumatic drug methotrexate (MTX) on Th1 and Th2 immune responses in mice. For this investigation, mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freund's adjuvant (day 0). Varying doses of MTX were orally administered daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-gamma (IFN-gamma) as indicators of Th1 responses and anti-OVA IgG1 and interleukin-10 (IL-10) as those of Th2 responses were measured. The results showed that treatment with MTX was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. The anti-rheumatic drug inhibited both anti-OVA IgG2a and IgG1 production, although the inhibitory effect of MTX on the antigen-specific IgG2a production appeared to be greater than that on IgG1 production. IFN-gamma, but not IL-10, secretion was markedly downregulated by MTX. Administration of MTX resulted in suppression of antigen (OVA)-induced arthritis in mice. The suppression of the joint inflammation by MTX was associated with inhibition of OVA-specific proliferative responses of spleen cells, anti-OVA IgG, IgG2a and IgG1 production, and IFN-gamma and IL-10 secretion, although more pronounced decreases in IgG2a and IFN-gamma were observed compared with those in IgG1 and IL-10 in MTX-treated mice. These results indicate that MTX appears to suppress Th1 and, to a lesser extent, Th2 immune responses and its anti-arthritic effect on human rheumatoid arthritis might be at least in part explained by down-regulation of Th1 responses involved in the disease.

Administration, Oral↗

PubMed2003

Isolation of nitrophenols from diesel exhaust particles (DEP) as vasodilatation compounds.

The compounds in diesel exhaust particles (DEP) that are responsible for vasodilatation were isolated and characterized for the first time. From benzene extract of DEP, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenl and 4-nitrophenol were isolated, and their vasodilatation activities were confirmed. 3-methyl-4-nitrophenol caused dilatation of rat thoracic artery, and the other two nitrophenols, also showed vasodilatation activities.

Air Pollutants↗

PubMed2002

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.

Aspartate Aminotransferases↗