[Complex cutaneous leishmaniasis with bone involvement]. / Komplexe kutane Leishmaniose mit ossärer Beteiligung.

We report an immunocompromised patient with a complex cutaneous leishmaniasis (CL) who suffered from singular bone involvement of the little left toe due to Leishmania (L.) infantum infection. The diagnosis was confirmed by positive polymerase chain reaction (PCR) testing in skin and bone tissue. The patient was successfully treated with miltefosine. In immunocompromised patients with CL, extracutaneous manifestations should always be ruled out. This is the first case report describing osseous involvement in CL due to Leishmania infantum.

[Medical and psychological aspects of the treatment of connatal dacryostenosis : Parental evaluation of their own and their child's stress]. / Medizinpsychologische Aspekte bei der Behandlung kindlicher Tränenabflussstörungen : Elterliche Beurteilung eigenen und kindlichen Stresserlebens.

BACKGROUND: Lacrimal probing and syringing for connatal dacryostenosis can be performed under local (LA) and general anesthesia (GA). In cases of invasive medical procedures, pain and anxiety can be distressing for children and their parents. MATERIALS AND METHODS: Using questionnaires (n = 65), parents were asked to evaluate their own stress and that of their child during lacrimal probing and syringing. Analyzing different subgroups, the impact of the kind of anesthesia (LA vs. GA), trust in medical treatment, therapeutic success, prior experiences with GA, parental educational level, age of parents and children, number of children, and time between the intervention and the interview on the stress was examined. Stress level was evaluated on a scale from 1 (no stress) to 10 (maximal stress). RESULTS: Mean children's age was 8.5 ± 7.42 months. Mean age of the parents was 30.8 ± 6.17 years. Treating children under LA, parents reported moderate to severe stress levels for themselves (mean, M = 7.15) and for their children (M = 7.82). Children's stress levels were significantly higher when the treatment was performed under LA (n = 47; M = 7.34) in comparison to GA (n = 18; M = 6.06; p < 0.05). Parents having two or more children reported significantly lower stress levels than those with only one child. Furthermore, prior experiences with GA led to significantly higher parental stress levels when their children were treated under GA. Other factors did not show any impact on parent's and children's stress levels. CONCLUSIONS: Parents might have been influenced by hearing the children's reaction (e. g., crying) during the intervention under LA. Nevertheless, parents had a higher acceptance of this type of intervention (LA) in comparison to GA. This effect was even stronger among parents with prior experience of GA.

Oncological results of oncoplastic breast-conserving surgery: Long term follow-up of a large series at a single institution: A matched-cohort analysis.

PURPOSE: Oncoplastic surgery is a well-established discipline that combines conserving treatment for breast cancer with immediate plastic reconstruction. Although widely practiced, the oncologic outcomes of this combined approach are reported only in small series. The aim of the present paper is to assess the safety of oncoplastic surgery for invasive primary breast cancer. METHODS: We compared 454 consecutive patients who underwent an oncoplastic approach between 2000 and 2008 for primary invasive breast tumors (study group) with twice the number of patients who received conservation alone in the same interval time (control group). Disease free survival and overall survival were estimated using the Kaplan-Meier method. The log-rank test was used to assess differences between groups. RESULTS: The median follow-up was 7.2 years. The overall survival is similar within the two groups, being 91.4% and 91.3% at 10-yr in the study group and in the control group respectively. The disease free survival is slightly lower in the oncoplastic group (69 vs.73.1% at 10-yr). The difference is not statistically significant. DISCUSSION: We have compared a large series of primary breast cancer patients that have undergone oncoplastic surgery (454) with a control group (908) and they were followed for a prolonged period of time. It provides the best available evidence to suggest that oncoplastic surgery is a safe and reliable treatment option for the managing of invasive breast cancer.

Incidence of bleeding in 8172 percutaneous ultrasound-guided intraabdominal diagnostic and therapeutic interventions - results of the prospective multicenter DEGUM interventional ultrasound study (PIUS study).

PURPOSE: To analyse the incidence of bleeding after percutaneous ultrasound guided diagnostic and therapeutic intraabdominal interventions in a prospective multicentre study (DEGUM percutaneous interventional ultrasound study). MATERIALS AND METHODS: Within a time period of 2 years diagnostic and therapeutic intraabdominal interventions (with the exclusion of ascites paracentesis) performed percutaneously under continuous ultrasound (US) guidance were prospectively assessed using a pseudonymized standardized web site entry form. Number and type of intervention, operator experience, patient characteristics, medication, lab data as well as technical aspects of the procedure and bleeding complications were analysed according to the interventional radiology standards. RESULTS: 8172 US-guided intraabdominal interventions (liver n = 5903; pancreas n = 501, kidney n = 434, lymph node = 272, biliary system n = 153, spleen n = 63, other abdominal organs and extra-organic targets n = 999) were analysed in 30 hospitals. The majority were diagnostic biopsies including 1780 liver parenchyma, 3400 focal liver lesions and 404 pancreatic lesions. 7525 interventions (92.1 %) were performed in hospitalized patients (mean age 62.6 years). Most operators were highly experienced in US-guided interventions (> 500 interventions prior to the study n = 5729; 70.1 %). Sedation was administered in 1131 patients (13.8 %). Needle diameter was ≥ 1 mm in 7162 punctures (87.9 %) with main focus on core needle biopsies (18 G, n = 4185). Clinically relevant bleeding complications with need of transfusion (0.4 %), surgical bleeding control (0.1 %) and radiological coiling (0.05 %) were very rare. Bleeding complications with fatal outcome occurred in four patients (0.05 %). The frequency of major bleeding complications was significantly higher in patients with an INR > 1.5 (p < 0.001) and patients taking a medication potentially interfering with platelet function or plasmatic coagulation (p < 0.0333). CONCLUSION: This prospective multicentre study confirms the broad spectrum of percutaneous US-guided intraabdominal interventions. However diagnostic liver biopsies dominate with the use of core needle biopsies (18 G). Percutaneous US-guided interventions performed by experienced sonographers are associated with a low bleeding risk. Major bleeding complications are very rare. A pre-interventional INR < 1.5 and individual medication risk assessment are recommended.

Estrategias para mejorar la sobrevida del prematuro extremo: [revisión] / Strategies to improve survival in extreme premature birth: [review]

Introduction: Biomedical advances have not decreased the number of premature deliveries. Newborns under 32 weeks constitute the most significant portion of infant mortality in developed countries. Objective: a literature review that identifies risk factors associated to premature labor and highlight strategies that increase survival. Results: Prenatal factor associated to early delivery include Black descent, maternal age, history of premature delivery, socioeconomic stratus, multiple deliveries and infections. Mortality is increased with earlier gestational age and lower fetal weight. Factors associated to increased survival include area of residence and place of birth, uterine transfer, variable levels of neonatal care, use of prenatal corticosteroids to accelerate pulmonary maturation, proactive management during childbirth, use of artificial surfactant and avoid use of steroids after delivery. Neonatal networks improve survival by potentiating best practices. Conclusions: Survival among these patients is enhanced by identification of risk factors for early delivery, knowing strategies that improve survival and planning for delivery in sites where best practices are offered.

La ciencia biomédica no ha logrado disminuir la incidencia de partos prematuros. Los RN menores de 32 semanas, constituyen el principal determinante de la mortalidad infantil en los países más desarrollados. Los objetivos de esta revisión son identificar a través de la literatura médica los factores de riesgo asociados a parto prematuro y destacar las estrategias desarrolladas para mejorar la sobrevida de este grupo etario. Resultados: Entre los factores prenatales asociados a prematurez destaca la raza negra, la edad materna, historia de parto prematuro previo, el nivel socioeconómico, embarazos múltiples e infecciones. Constituyen factores de riesgo asociados a mortalidad neonatal, tener menor edad gestacional y peso, ser PEG. Dentro de las estrategias para mejorar la sobrevida destaca la regionalización, el traslado in útero, establecer diferentes niveles de atención en el cuidado neonatal, el uso corticoides pre natal para acelerar la madurez pulmonar, tomar una conducta proactiva al nacer, el uso de surfactante artificial y evitar tratamientos con corticoides después de nacer. Las redes neonatales permiten mejorar la sobrevida potenciando las mejores prácticas médicas. El lugar en que se nace es importante en la sobrevida del recién nacido, Conclusión: Identificar los factores de riesgo de parto prematuro y conocer las estrategias que mejoran la sobrevida de los RN menores de 32 semanas permite planificar el lugar de nacimiento y ofrecer las mejores prácticas clínicas destinadas a reducir la mortalidad de este grupo.

New function of the amino group of thiamine diphosphate in thiamine catalysis.

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase--4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

Luxación congénita de cadera: displasia evolutiva de la cadera (DEC) / Congenital dislocation of the hip: developmental displasia of the hip

The change of denomination of congenital hip luxation for evolutionary hip displasia is defined and explained, it incluyes luxation, subluxation and hip instability. The frequencies of this pathology in the Clinical Hospital of the University of Chile is reported. The finds of significant major frequency in female newborn children, breech presentation and left hip are communicated. Thefamily base of this pathology is confirmed. The recommendations of the experts’ Committee of the American Academy of pediatrics and those of the health department of Chile are given. It is emphasized that the diagnosis must be as precocious as possible and that the best method of diagnosis is Ortolani’s or Barlow maneuver, done by a professional of experience. It is indicated the oportunity in which the ultrasound scan and the hip X-ray must be done, also the recommended treatment. The use of the double diaper is scorned and its possible sequels arecommented.

Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: [formula: see text] During the first stage, an inactive intermediate complex (TK...ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* - ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hübner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 - 4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k(-1): in the presence of the substrate, its value is decreased several fold, whereas K(d) and k(+1) remain unchanged.

Impacto economico de la prematurez y las malformaciones congénitas sobre el costo de la atención neonatal

Objetivos: Calcular los costos de la atención neonatal de recién nacidos prematuros y en portadores de malformaciones congénitas mayores compatibles con la vida. Pacientes y Método: Estudio retrospectivo efectuado en el Departamento de Ginecología y Obstetricia del Hospital Clínico de la Universidad de Chile, en 82 recién nacidos menores de 34 semanas de gestación y en 14 con malformaciones congénitas mayores, de más de 37 semanas de gestación, compatibles con la vida, atendidos entre enero y diciembre de 2004. Resultados evaluados son los costos de la atención neonatal subdivididos en componentes. Resultados: El costo promedio de la atención neonatal en recién nacidos menores de 34 semanas fue igual a $2.519.508, en menores de 32 semanas igual a $3.766.999, en menores de 1500 gramos igual a $12.017.650 y en portadores de malformaciones congénitas mayores compatibles con la vida de $30.967.180. El día cama representa el componente más significativo dentro cada paquete con más del 60 por ciento del costo promedio. Conclusiones: El costo de la atención neonatal de prematuros menores de 34 semanas o portadores de malformaciones congénitas mayores compatibles con la vida es mayor al contemplado en los paquetes de prestaciones a todo evento, representando el día cama su componente más significativo.

Objective: To calculate the cost involved in the neonatal care of premature or live born babies carriers of mayor congenital abnormalities compatible with life. Patients and method: Retrospective study who analyzed 82 premature live born of less than 34 weeks and 14 live born carriers of mayor congenital abnormalities compatible with life, from January to December 2004, at the Maternity Ward from the University of Chile Clinical Hospital. The outcome measures were the neonatal care average cost package subdivided by components. The cost was expressed in chilean currency. Results: Neonatal care average cost was $2.519.508 in live born of less than 34 weeks, $3.766.999 in less than 32 weeks, $12.017.650 in less than 1500 grams babies and $30.967.180 in carriers of mayor congenital abnormalities. In bed day cost represents the most significant component from the package, representing more than 60 percent of its total cost. Conclusions: Neonatal care cost of premature live born of less than 34 weeks or carriers of mayor congenital abnormalities compatible with life is higher than the maximum cost considered in the every-event health packages, representing in bed day its most significant component.

Effect of transketolase substrates on holoenzyme reconstitution and stability.

The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.

Hepatic arterial doppler sonography in patients with cirrhosis and controls: observer and equipment variability with use of the ultrasonic contrast agent SHU 508A.

PURPOSE: The aims of this study on hepatic arterial Doppler sonography were to ascertain interobserver and interequipment variability, to investigate any potential artificial influence of the ultrasonic contrast agent on the Doppler measurements and to compare the results in healthy and cirrhotic subjects. METHODS: Doppler sonography of the left hepatic artery was performed in nine healthy and nine cirrhotic subjects by three independent observers using three different devices. Continuous infusion of the ultrasonic contrast agent SHU 508A and placebo were administered in a double blind fashion. Systolic, mean and end diastolic peak velocities as well as resistive and pulsatility indices were measured. RESULTS: Equipment associated variances (5.8 - 12.7 %) of the five Doppler parameters were greater than interobserver variances (0.3 - 3.6 %). No significant differences were observed between the velocities using ultrasonic contrast agent and placebo. Systolic (65.9 +/- 3.6 vs. 47.7 +/- 4.2 cm/s mean +/- SE, p = 0.02) and mean peak velocity (35.4 +/- 1.6 vs. 24.5 +/- 1.8 cm/s, p = 0.007) were significantly higher in cirrhotic than in healthy subjects whereas the resistive and pulsatility indices were not different. CONCLUSIONS: Doppler sonography of the left hepatic artery performed by various observers is reproducible as long as the same device is used. Under clinical conditions, velocities are correctly measured with the use of ultrasonic contrast agent and are elevated in patients with cirrhosis.

Effect of humidity on the supramolecular structure of cotton, studied by quantitative spin probing.

The effect of water content on the physicochemical properties of the amorphous regions in cotton were investigated by measuring the electron paramagnetic resonance (EPR) of TEMPOL nitroxide radicals, deposited in cotton at different loadings, as a function of the relative humidity (RH) and temperature. Three different components contribute differently to the experimental EPR spectra, corresponding to (a) mobile radicals absorbed in the bulk amorphous region, (b) slow moving radicals adsorbed on the crystallite surfaces in cotton, and (c) aggregated radicals. These components were analyzed by means of computer-aided simulations of the line shapes and simplified line width methods. Polarity and mobility parameters were extracted from the analysis of the spectra. For all loadings and temperatures, the polarity suddenly dropped when the water content fell below approximately 3 wt %, i.e., when water was removed from the bulk amorphous regions. At the lowest loading (2 x 10(-5) mol kg(-1)), the spectra were independent of the RH, and only mobile radicals were observed. At intermediate loading (10(-4)-10(-3) mol kg(-1)) both mobile (fast) and adsorbed (slow) moving radicals were present, the fraction of which depended on the RH. The mobility of the adsorbed and mobile radical signals was smaller at higher loadings, indicating microdomains of different character. The temperature dependence of the rotational correlation times provided the activation energies, which were much lower than in liquids. An equilibrium exists between the mobile and the adsorbed radicals. The temperature dependence of the equilibrium constant, K, gave the enthalpy and the entropy of the adsorption process. At low RH, the enthalpy and the entropy values indicated a simple adsorption process. At 10(-3) mol kg(-1), the values were independent of the RH, but at low loadings the values increased with the increase in the RH, which suggested a displacement of adsorbed water by the radicals at high water content. At loadings above 10(-3) mol kg(-1), signals from radicals strongly interacting via spin exchange were observed, which are assigned to aggregated radicals; simulation of the spectra gave an activation energy of 13 kJ mol(-1) for the spin exchange process. These effects are rationalized on the basis of microdomains of different character within cotton, reflecting the variation in pore sizes (0.5-8 nm) and the relaxation behavior of the cellulose chains.

Mortalidad y factores de riesgo asociados a depresión cardio-respiratoria prolongada al nacer / Mortality and associated factors of risk to prolonged cardiorespiratory depression when being born

Existe mayor mortalidad y morbilidad entre los recién nacidos (RN) que mantienen una depresión cardiorrespiratoria a los cinco minutos de vida a pesar de las maniobras de reanimación. Objetivos: individualizar los factores de riesgo que se asocian a esta condición. Pacientes y método: se estudian los recién nacidos con Apgar menor o igual a tres en el período de enero 2003 a mayo del 2004. Se separan en dos subgrupos. Aquellos que se recuperan con maniobras de reanimación y aquellos RN que mantienen la depresión cardiorrespiratoria a los cinco minutos . Las variables estudiadas fueron peso de nacimiento, edad gestacional (EG) en semanas, la relación entre EG y peso, edad materna, multiparidad, morbilidad materna, factores fetales y ovulares. Resultados. De un total de 1705 RN, 64 RN presentan Apgar menor o igual a tres al minuto de vida. 22 RN ( 34,4 por ciento) no mejoran con las maniobras se reanimación a los 5 minutos. De las variables estudiadas son estadísticamente significativas para mantener una depresión severa al nacer, el extremo bajo peso de nacimiento, edad gestacional baja y la condición de ser pequeño para la edad gestacional (PEG). La mortalidad en este grupo es significativamentemayor. Conclusión Los RN que mantienen depresión a los cinco minutos tienen un peso de nacimiento y EG significativamente menor que aquellos que se recuperan con las maniobras de reanimación y tienen mortalidad significativamente mas alta.

A high rate of morbidity and mortality is present among newborns that maintain a cardiorespiratorydepression at five minutes of life in spite of resuscitation maneuvers. Therefore, it is important to know whichrisk factors are associated with this condition. In the time period between January 1, 2003 and May 1, 2004 there was a total of 1,705 alive newborns. Of these 64 (3,75 percent)had an Apgar score of three or less at a minute of life. Among these there were 22 newborns (34,4 percent) that did not improve with resuscitation maneuvers at 5 minutes of life. Amongst the studiedfactors stand out that newborns that presented prolonged depression had birth weight and gestational age significantly lower than those who recover with resuscitation maneuvers. The newborns with cardiorespiratory depression at 5 minutes of life havealmost twice the mortality rate of those born depressed but that improve before 5 minutes of life.

Malformaciones congénicas del sistema digestivo: maternidad Hospital Clínico de la Universidad de Chile: período 1991-2001 / Frequency of digestive system congenital malformations detected at the University of Chile hospital in the period 1991-2001

There were 26,617 births attended at the University of Chile Clinical Hospital, of which 0.63 percent were stillborn. A frequency of malformations of 7.2 per born alive and of 22.1 per stillborn was detected in this population. Aim: To report the frequency of digestive system congenital malformations in this population. Material and methods: Analysis of data from the births that occurred at the University of Chile Hospital, that was gathered using codified form for the Latin American Collaborative Study for Congenital Malformations. Results: Ninety seven digestive congenital malformations were detected, with a rate of 26.4 per 10,000 born alive and 12.2 per 10,000 stillborn. Diaphragmatic hernia was the most frequent malformation found, followed by imperforated anus, onphalocele and esophageal atresia. There was a secular increase in the frequency of these malformations. Conclusions: The frequency of digestive system malformations is higher than in the rest of hospitals participating in the Latin American Collaborative Study for Congenital Malformations

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase.

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.

Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis.

Recently, we demonstrated a strong upregulation of activin expression after skin injury. Furthermore, overexpression of this transforming growth factor beta family member in the skin of transgenic mice caused dermal fibrosis, epidermal hyperthickening and enhanced wound repair. However, the role of endogenous activin in wound healing has not been determined. To address this question we overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice. These animals were born with open eyes, and the adult mice had larger ears, longer tails and reduced body weight compared with non-transgenic littermates. Their skin was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was significantly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller than in control animals. These results implicate an important function of endogenous activin in the control of wound repair and scar formation.

Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions.

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Consequences of a modified putative substrate-activation site on catalysis by yeast pyruvate decarboxylase.

Earlier, it had been proposed in the laboratories at Halle that a cysteine residue is responsible for the hysteretic substrate activation behavior of yeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C221 as the site where substrate is bound to transmit the information to H92, to E91, to W412, and finally to the active center thiamin diphosphate. According to steady-state kinetic assays, the C221A/C222A variant is no longer subject to substrate activation yet is still a well-functioning enzyme. Several further experiments are reported on this variant: (1) The variant exhibits lag phases in the product formation progress curves, which can be attributed to a unimolecular step in the pre-steady-state stage of catalysis. (2) The rate of exchange with solvent deuterium of the thiamin diphosphate C2H atom is slowed by a factor of 2 compared to the wild-type enzyme, suggesting that the reduced activity that results from the substitutions some 20 A from the active center is also seen in the first key step of the reaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V(max)/K(m) (0.62), and small but normal on V(max) (1.26), virtually ruling out residue C221 as being responsible for the inverse effects reported for the wild-type enzyme at low substrate concentrations. The solvent kinetic isotope effects are compared to those on two related enzymes not subject to substrate activation, Zymomonas mobilis pyruvate decarboxylase and benzoylformate decarboxylase.