Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure

A gyulladásos bélbetegségek extraintestinalis manifesztációi és kezelésük = Extraintestinal manifestations of inflammatory bowel diseases and their management

A colitis ulcerosa és a Crohn-betegség extraintestinalis manifesztációi olyan egyéb szervi tünetek, amelyek a szisztémás gyulladásos folyamat által okozott változatos szervkárosodások következményei. Elsődlegesnek a bél megbetegedése látszik, de bizonyos genetikai hajlam esetén az immunreguláció zavarának következtében egyéb szervek gyulladása is megjelenhet a bélgyulladással egyidejűleg, azt megelőzően vagy akár a nyugalmi állapotban is. Az extraintestinalis manifesztációk lehetőség szerint elkülönítendők a gyulladásos bélbetegség szövődményeitől és a gyógyszeres kezelés mellékhatásaitól. Leggyakoribb a csontok és ízületek, a bőr, a szem, máj és epeutak érintettsége, de előfordulhat a véralvadási rendszer, tüdő, vese és az idegrendszer megbetegedése is. Kezelésüket befolyásolja a bélbetegség aktivitásával való összefüggésük. Az aktivitástól független manifesztációk kezelésében a speciális beavatkozásokon túl legtöbbször tartós gyulladásgátló vagy immunmoduláns kezelés is szükséges. A különböző szintű evidenciákat szolgáltató közlemények alátámasztják a biológiai terápia hatékonyságát gyulladásos bélbetegségek különböző extraintestinalis manifesztációiban is. A betegek gondozása, követése többszakmás feladat. Orv. Hetil., 2011, 152, 663–671. | Extraintestinal manifestations of ulcerative colitis and Crohn’s disease are symptoms of variegated organ injuries due to systemic inflammatory process. Bowel disease seems to be primary but upon genetic disposition disturbed immune regulation may result in inflammation of other organs that may appear simultaneously, precede bowel disease or follow it in the quiescent phase. As far as possible extraintestinal manifestations have to be distinguished from the complications of the intestinal inflammation and also from side effects of drugs for its treatment. Involvement of bones and joints, skin, eyes, liver and biliary tract is more common but disease of blood coagulation system, lung, kidneys and nervous system may also appear. Treatment of extraintestinal manifestations is influenced by their association with the activity of the bowel disease. Beyond some specific interventions, to achieve improvement of manifestations independent from activity long lasting anti-inflammatory therapy or immunomodulation is almost always needed. Clinical evidence of different levels from reports support the favorable effect of biologic therapy in different extraintestinal manifestations of inflammatory bowel diseases as well. Care and follow-up of patients is a multidisciplinary task. Orv. Hetil., 2011, 152, 663–671

A gyomor micropneumatosisa: egy ritka endoszkópos kórkép = Gastric micropneumatosis: a rare endoscopic entity

A ritkán diagnosztizált gyomor micropneumatosist a nyálkahártyában felszaporodó levegő okozza. Nagyobb százalékban észlelhető Helicobacter pylori pozitív atrophiás gastritisben, más esetben pneumatosishoz társulhat, melynek hátterében gázképző baktériumok, illetve nem infekciózus okok állhatnak. A kórképet egy 69 éves hipertóniás, generalizált arteriosclerosisban szenvedő, agyi infarktuson átesett, clopidogrelt szedő, szociális otthonban élő, savhaematinos hányások, melaena miatt felvételre került férfi betegben észleltük. Felső panendoszkópia során a kifejezetten lobos mucosájú gyomorban, a fundus-corpus határon, a hátsó falon tenyérnyi, térképszerű képet mutató, helyenként kissé előemelkedő, sárgás foltokat tartalmazó nyálkahártya-területet észleltünk. A szövettani vizsgálat során a corpusmirigyek között észlelt 10–20 µm átmérőjű, epithelium béléssel nem rendelkező lyukak alapján a gyomor micropneumatosisára következtettünk. Esetünkben feltehetőleg az ischaemia okozta csökkent mucosa integritásnak és az ismétlődő hányásoknak lehetett szerepe, mivel az utóbbi megszűnte után a 3 nap múlva elvégzett kontroll-endoszkópia és szövettani vizsgálat eltérést már nem mutatott. A szerzők a micropneumatosis ventriculi általuk észlelt képének ismertetésével kívánják felhívni a figyelmet e ritka elváltozásra, és a háttérben rejlő lehetséges okokat is tárgyalják. The rarely described gastric micropneumatosis is caused by accumulating air in the mucus membrane. It may associate mainly with Helicobacter pylori positive atrophic gastritis or with pneumatosis due to gas producing bacteria or non-infectious causes. In our case the disorder was diagnosed in a clopidogrel taking 69 years old patient, living in a twilight home, with history of hypertension, general arteriosclerosis, cerebral infarction, who was admitted to our department because of melaena and hematemesis. Upper endoscopy revealed a map-like, slightly raised, palm-sized area with yellowish spots on the posterior wall of the markedly inflamed stomach at the level of fundus – corpus border. Based on histology findings, that showed empty spaces among corpus glands, 10–20 µm in diameter, with no epithelial lining, the diagnosis was gastric micropneumatosis. The impaired mucosal integrity caused by mesenteric ischaemia and repeated vomiting were supposed as possible etiological factors, because 72 hours after the patient had stopped vomiting, control endoscopy showed regression, and the empty spaces were no more detectable in the histology samples. By this case report the authors would like to draw attention to a seldom diagnosed gastric injury, and discuss the factors that may predispose the development of gastric micropneumatosis

Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1–13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme–substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure