ABSTRACT
Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.
Subject(s)
Molecular Dynamics Simulation , Proteins , Crystallography, X-Ray , Proteins/chemistry , Catalysis , Protein Conformation , HydrolasesABSTRACT
Abstract:The Indian hill trout cyprinid, Barilius bendelisis is a member of family Cyprinidae that dwells in shallow, cold, and clear water. In this study, growth parameters and reproductive biology of Indian hill trout, Barilius bendelisis from river Gaula, Central Himalaya region, India, were studied. The length-frequency data were grouped sex wise and were analyzed to determine the growth and mortality parameters using the computer software programme, FAO-ICLARM Stock Assessment Tool (FISAT II). Altogether, 501 individuals were collected from river Gaula (November 2013-October 2014) and were preserved in formalin for further analysis. The results showed that the female outnumbered the male population. The minimum GSI of females was observed in the month of October (4.93 ± 0.26) and for males in the month of June and July (0.093 ± 0.12), whereas, the maximum value was in the month of April for both females (13.47 ± 0.52) and males (1.21 ± 0.12). Fluctuation in GSI values had a bimodal pattern showing two peaks during March-May and August-September in both the sexes, indicating the common spawning period of fish. The slope of regression showed the negative allometric growth for both males and females (b= 2.65 for male and b= 2.5 for female). A significant relationship between length and weight was observed in the present study (p < 0.05). The ELEFAN-I estimated L∞ and K of the von Bertalanffy growth factor for males (17.33 cm and 0.310 per year), females (17.33 cm and 0.3 per year) and pooled sexes (17.33 cm and 0.240 per year). The results indicated that Barilius bendelisis is a small sized fish having negative allometric growth that spawns twice a year. Thus, the present study on biological traits represents the baseline information for effective production, conservation and restoration planning. Rev. Biol. Trop. 64 (3): 1287-1295. Epub 2016 September 01.
ResumenBarilius bendelisis es un miembro de la familia Cyprinidae que habita en agua superficial, fría y clara. En esta investigación se estudiaron los parámetros de crecimiento y la biología reproductiva de Barilius bendelisism en el río Gaula, Himalaya Central, India. Los datos talla-frecuencia fueron agrupados por sexo, y analizados para determinar parámetros de crecimiento y mortalidad usando el programa de computador, FAO-ICLARM Stock Asseessment Toll (FISAT II). En total, 501 individuos fueron recolectados en el río Gaula (Noviembre 2013-Octubre 2014) y fueron preservados en formalina para futuros análisis. Los resultados mostraron que la población de hembras supera en número la población de machos. El GSI mínimo en hembras fue observado en octubre (4.93 ± 0.26) y en machos en Junio y Julio (0.093 ± 0.12), mientras, el valor máximo en abril para ambos, hembras (13.47 ± 0.52) y machos (1.21 ± 0.12). La fluctuación en los valores del GSI tenía un patrón bimodal mostrando dos picos durante Marzo-Macho y Agosto-Septiembre en ambos sexos, indicando el periodo de desove común del pez. La pendiente de regresión muestra crecimiento alométrico negativo tanto para machos como para hembras (b= 2.65 para machos y b= 2.5 para hembras). Se observó una significativa relación entre talla y peso en el presente estudio (p < 0.05). El ELEFAN-I estimó L∞ y K del factor de crecimiento von Bertalanffy para machos (17. 33 cm y 0.310 por año), hembras (17.33 cm y 0.3 por año) y sexos combinados (17.33 cm y 0.240 por año). Los resultados indicaron que Barilius bendelisis es un pez de talla pequeña con un crecimiento alométrico negativo que desova dos veces al año. Además, el presente estudio de rasgos biológicos representa la información de base para la efectiva producción, conservación y planeamiento de la restauración.