Biomedical subjects

B H Oh

Publications and source records attributed to B H Oh.

At least 37 records · Page 2Linked to original sources

PubMed2001

Effect of hypercholesterolemia on macrophage infiltration after balloon injury to rabbit iliac artery.

Both hypercholesterolemia and vascular injury have been reported to induce macrophage infiltration, but their combined effect and the mechanism by which hypercholesterolemia enhances the infiltration remain to be clarified in vivo. To evaluate the effect of hypercholesterolemia on macrophage infiltration after vascular injury, the iliac arteries of hypercholesterolemic (HC) and normocholesterolemic (NC) rabbits were examined 2h, 1 day, 3 days, 7 days, and 14 days after balloon injury using immunohistochemical staining for macrophages, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1. Nuclear factor kappa-B (NF-kappaB) activation was also evaluated in fresh frozen iliac arteries using the electrophoretic mobility shift assay method. The fundamental difference between HC and NC was the amount of macrophage infiltration seen in HC from 7 days after balloon injury. Two out of 4 HC iliac arteries on the 7th day, and 3 out of 4 HC iliac arteries on the 14th day were positively stained with ICAM-1 in regenerated endothelium and neointima, whereas there were no positively stained NC iliac arteries. Neither HC nor NC tissues showed positive staining with VCAM-1. NF-kappaB was activated in HC 7 and 14 days after balloon injury, but not in NC. In conclusion, in vivo hypercholesterolemia induces macrophage infiltration after balloon injury and it is mediated by increased NF-kappaB activation promoting ICAM-1 expression.

Animals↗

PubMed2000

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes.

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.

Binding Sites↗

PubMed2000

Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases.

Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by carrying out a detailed investigation of the enzymatic reaction mechanism. This investigation included the X-ray determination of 15 crystal structures of two homologous enzymes in free and complexed states (with inhibitors) and extensive ab initio calculations of the interactions between the active sites and the reaction intermediates. The catalytic residues, through short strong hydrogen bonds, play the role of charge buffer to stabilize the negative charge built up on the intermediates in the course of the reaction. The hydrogen bond distances in the intermediate analogues are found to be about 0.2 A shorter in the product analogues both experimentally and theoretically.

Aspartic Acid↗

PubMed2000

Crystal structure of TRAIL-DR5 complex identifies a critical role of the unique frame insertion in conferring recognition specificity.

TRAIL is a cytokine that induces apoptosis in a wide variety of tumor cells but rarely in normal cells. It contains an extraordinarily elongated loop because of an unique insertion of 12-16 amino acids compared with the other members of tumor necrosis factor family. Biological implication of the frame insertion has not been clarified. We have determined the crystal structure of TRAIL in a complex with the extracellular domain of death receptor DR5 at 2.2 A resolution. The structure reveals extensive contacts between the elongated loop and DR5 in an interaction mode that would not be allowed without the frame insertion. These interactions are missing in the structures of the complex determined by others recently. This observation, along with structure-inspired deletion analysis, identifies the critical role of the frame insertion as a molecular strategy conferring specificity upon the recognition of cognate receptors. The structure also suggests that a built-in flexibility of the tumor necrosis factor receptor family members is likely to play a general and important role in the binding and recognition of tumor necrosis factor family members.

Binding Sites↗

PubMed2000

Role of the glutamate 332 residue in the transglycosylation activity of ThermusMaltogenic amylase.

A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in alpha-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on alpha-(1,3)-, alpha-(1,4)-, and alpha-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

Acarbose↗

PubMed2000

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family.

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

Amino Acid Sequence↗

PubMed2000

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.

Amino Acid Substitution↗

PubMed2000

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.

Asparagine↗

PubMed2000

Synergistic effect of angiotensin-converting enzyme and angiotensinogen gene on cardiac hypertrophy.

AIMS: There are controversies concerning the association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism with left ventricular hypertrophy (LVH), and the unclear association between angiotensinogen (ATG) M235T polymorphism and LVH. We investigated both the separate and interactive effects of these two genes on LVH in patients (N=396) with cardiovascular disease and normal healthy volunteers (N=133). RESULTS: Frequency of DD genotype of ACE gene was significantly (P<0.05) higher in patients with LVH than patients without LVH or normal controls. Frequency of IT genotype of ATG gene in patients with LVH was significantly (P<0.01) greater than that in normal controls or marginally (P=0.1) higher than that in patients without LVH. These findings were also observed in normotensive patients and normal controls after excluding hypertensive patients. Only in patient group, the frequency of DD genotype in the highest quartile of LVMI was significantly greater than that in the lowest quartile (P<0.05). The higher tendency of TT genotype in the highest quartile patients compared with that in the lowest, did not reach statistical significance. In combined genotype analysis, there was a remarkable difference in LVMI between the two extreme double homozygotes only in patient group (156+/-25 versus 109+/-25 g/m2 for TT+DD versus MM+II) (P<0.01). In ANCOVA, the interaction term composed of ACE and ATG genotype was a significant independent variable for LVMI only in the male patient group (P<0.01). CONCLUSION: The D-allele of ACE and T-allele of ATG gene exert a synergistic effect on cardiac hypertrophy in male patients with cardiovascular diseases, but not in normal healthy population.

Adult↗

PubMed2000

Limited performance of quantitative assessment of myocardial function by thallium-201 gated myocardial single-photon emission tomography.

We investigated the reproducibility between thallium-201 and technetium-99m methoxyisobutylisonitrile (MIBI) gated single-photon emission tomography (SPET) for the assessment of indices of myocardial function such as end-diastolic and end-systolic volume (EDV, ESV), ejection fraction (EF) and wall motion. Rest 201Tl (111 MBq) gated SPET was sequentially performed twice in 20 patients. Rest 201Tl gated SPET and rest 99mTc-MIBI (370 MBq) gated SPET were performed 24 h apart in 40 patients. Wall motion was graded using the surface display of the Cedars quantitative gated SPET (QGS) software. EDV, ESV and EF were also measured using the QGS software. The reproducibility of functional assessment on rest 201Tl gated SPET was compared with that on 99mTc-MIBI gated SPET, and also with that between 201Tl gated SPET and 99mTc-MIBI gated SPET performed on the next day. The two standard deviation (2 SD) values for EDV, ESV and EF on the Bland-Altman plot were 29 ml, 19 ml and 12%, respectively, on repeated 201Tl gated SPET, compared with 14 ml, 11 ml and 5.3% on repeated 99mTc-MIBI gated SPET. The correlations were good (r=0.96, 0.97 and 0.87) between the two measurements of EDV, ESV and EF on repeated rest studies with 201Tl and 99mTc-MIBI gated SPET. However, Bland-Altman analysis revealed that the 2 SD values between the two measurements were 31 ml, 23 ml and 12%. We were able to score the wall motion in all cases using the 3D surface display of the QGS on 201Tl gated SPET. The kappa value of the wall motion grade on the repeated 201Tl study was 0.35, while that of the wall motion grade on the repeated 99mTc-MIBI study was 0.76. The kappa value was 0.49 for grading of wall motion on repeated rest studies with 201Tl and 99mTc-MIBI. In conclusion, QGS helped determine EDV, ESV, EF and wall motion on 201Tl gated SPET. Because the EDV, ESV and EF were less reproducible on repeated 201Tl gated SPET or on 201Tl gated SPET and 99mTc-MIBI gated SPET on the next day than on repeated 99mTc-MIBI gated SPET, functional measurement on 201Tl gated SPET did not seem to be interchangeable with that on 99mTc-MIBI gated SPET.

Coronary Disease↗

PubMed2000

Analysis of mitochondrial DNA deletions in four chambers of failing human heart: hemodynamic stress, age, and disease are important factors.

Mitochondrial DNA (mtDNA) mutations are not only responsible for organ dysfunction due to inefficient energy production but also indicators of metabolic and functional stresses in the organ. To analyze the significance of deletion mutation in human myocardium, we screened the presence of two common deletions (7.4 kb from 8637-16084 nt, 5.0 kb from 8470-13477 nt) in four chambers using long-PCR, and using serial-dilution PCR, measured the amount of deleted mtDNA in normal heart (NL) of brain-dead victims of road accidents (n = 9, age = 10-59) and failing hearts (CHF) of patients who underwent heart transplantation (n = 24, age = 17-63). Frequency of both deletions was higher in ventricles (Vt) than in atria (At) (Vt:At = 25/33:12/33 for 7.4 kb, 19/33:6/33 for 5 kb) (p < 0.05), whereas it was the same in the right and left chambers. In ventricles, both deletions were more frequent among older persons (> 35 yrs) than in younger persons (< or = 35 yrs) (older:younger = 16/20:9/13 for 7.4 kb, 15/20:4/13 for 5 kb) (p < 0.05). In ventricles of failing heart, the 5-kb deletion was more frequent than in those of normal heart (CHF:NL = 17/24:2/9) (p < 0.05), whereas the 7.4-kb deletion was frequent both in failing and normal hearts (CHF:NL = 19/24:6/9). The association of mutation with aging or disease process observed in ventricles was not found in the atria. Although the amount of mutant mtDNA in the left ventricle tended to increase according to a disease process, it was small, at most 1.56% or 0.012% of total mtDNA for a 7.4- or 5-kb deletion, respectively. No deletion was found, however, in lymphocytes from any patient who underwent transplantation. In conclusion, deletion mutation of mtDNA is frequently, but in a small amount, found in the ventricle of older failing heart than in the atrium of younger normal heart. This suggests that hemodynamic stress, age, and disease are factors to induce mtDNA mutation that represents the indicator of stresses on the heart and might turn into a contributor of progressive heart failure under extreme conditions.

Adolescent↗

PubMed2000

Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states.

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as approximately 30 degrees C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.

6-Phytase↗

PubMed2000

Effects of red ginseng upon vascular endothelial function in patients with essential hypertension.

This study is to estimate the effect of Korean red ginseng on vascular endothelial cell dysfunction in patients with hypertension. Seventeen patients with hypertension who were divided into ginseng-treated (7) and non-treated (10) groups and 10 normotensive subjects were included. To assess the function of the vascular endothelial cell, changes of forearm blood flow to infusion of acetylcholine, sodium nitroprusside and bradykinin in incremental doses were measured by venous occlusion plethysmography. In the ginseng-treated hypertensive group, forearm blood flows at the highest dose of acetylcholine and bradykinin were significantly higher than those of the non-treated hypertensive group and were not different from those of the control group. In the case of sodium nitroprusside infusion, no significant differences were observed between the control, non-treated and treated groups. In conclusion, Korean red ginseng can improve the vascular endothelial dysfunction in patients with hypertension possibly through increasing synthesis of nitric oxide.

Acetylcholine↗

PubMed2000

Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling.

To verify that apoptosis is one of the possible mechanisms of neonatal vascular remodeling during the transition from fetal to neonatal circulation, we assayed for apoptosis and evaluated the expression of apoptosis-regulatory proteins in umbilical vessel versus ascending aorta, ductus arteriosus (DA) versus adjacent pulmonary artery and aorta, or aorta versus its branching arteries. Twenty-two umbilical cords (UCs), 6 DAs with adjacent aortas and pulmonary arteries, and 4 aortic arches with their branching great arteries were obtained from neonates. Smooth muscle cell (SMC) apoptosis in umbilical vessels was identified in all UCs. The expressions of Bax and Bcl-X were stronger in umbilical artery than in the neonatal aorta, but Bcl-2 was weak in both arteries in immunohistochemistry. In the immunoblot analysis of UCs, the expression of the proapoptotic short isoform of Bcl-X was stronger than in other tissue, and caspase-3 was selectively activated, whereas it was not in the other components of the cardiovascular system. In contrast, the expression patterns of the FasAg and Fas ligand were similar in umbilical artery and aorta. Regulation of Bcl-2 family proteins was also observed in other vascular sites at which SMCs undergo apoptosis on hemodynamic changes during birth, such as the DA and the branching points of the great arteries from the aortic arch. Apoptosis is involved in the regression of human umbilical vessels and the DA and in the remodeling of the branching great arteries during the neonatal period, when Bcl-2 family proteins are likely to play a key role.

Aorta↗

PubMed1999

Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.

Binding Sites↗

PubMed1999

Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B.

The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --> Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.

Alanine↗

PubMed1999

Crystal structure of a maltogenic amylase provides insights into a catalytic versatility.

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

Amino Acid Sequence↗

PubMed1999

Determination of the limited trypsinolysis pathways of tumor necrosis factor-alpha and its mutant by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

Amino Acid Sequence↗