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Peer promotion programs and social networks in Ghana: methods for monitoring and evaluating AIDS prevention and reproductive health programs among adolescents and young adults.

This article summarizes the observations and lessons learned regarding the application of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) prevention and reproductive health evaluation methodologies in the context of adolescent and young adult populations and discusses the use of peer network evaluation to understand the dynamics of peer promotion. To examine the interpersonal communication process of peer education, this study tested a new approach using multiple semistructured interviews and network analysis to collect data on 106 peer educators and 526 of their contacts. These evaluation activities were conducted at three sites in Ghana during April 1998, in both periurban and rural locations in both in-school and out-of-school settings. By evaluating the social networks of peer educators it was possible to gain a better understanding of the process of peer education in terms of (a) defining the composition of peer contacts, (b) identifying the social norms that play a critical role in youth decision making, and (c) observing the range of messages and services transmitted during peer education. The objective of this paper is to disseminate the experience of the Center for Education and Development of Population Activities (CEDPA) and Focus on Young Adult's cooperative development of evaluation methodologies for peer promotion and to highlight utilization of these methodologies in a case study in Ghana. The results will be discussed in terms of their possible implications for program managers, researchers, and international agencies.

Acquired Immunodeficiency Syndrome↗

Chromosomal localization and promoter analysis of the adenomatous polyposis coli binding protein RP1.

The EB1/RP1 family is a new protein family that is characterized by the ability of its members to serve as interacting partners for the adenomatous polyposis coli (APC) tumour suppressor protein and tubulin. Data obtained with highly conserved yeast homologues suggest that the EB1/RP1 protein family promotes cytoplasmic microtubule dynamics and contributes to the sensor mechanism controlling the cytokinesis checkpoint during mitosis. However, the precise function of this protein family in mammalian cells has not been elucidated so far and remains unclear. Here, we report on the genomic localization of the RP1 gene and the characterization of the corresponding promoter. The RP1 gene was found to be encoded on chromosome 18q21, a locus which is altered or deleted in up to 50% of all patients with colorectal cancer. Promoter analysis revealed that the RP1 gene is under the control of a strong promoter that was 10 times more active in mammalian cells when compared to SV40 promoter. Members of the cyclic AMP response element binding protein family (CREB1 and CREB2) could be identified as transcription factors binding specifically within the RP1 promoter sequence.

Base Sequence↗

Recruitment of distinct chromatin-modifying complexes by tamoxifen-complexed estrogen receptor at natural target gene promoters in vivo.

Tamoxifen, a breast cancer therapeutic, is a tissue-selective estrogen receptor modulator (SERM), which acts as an antiestrogen in the mammary tissue and displays estrogenic activity in other tissues such as bone and uterus. In order to understand the mechanisms underlying the antiestrogenic effect of this prototype SERM, we performed an analysis of the cofactors that interact with ER complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7. Employing chromatin immunoprecipitation (ChIP), we observed that treatment with OHT rapidly induces the binding of ERalpha to the E-responsive promoter regions of pS2 and c-myc genes. Promoter-bound OHT-complexed ERa coordinately recruited the components of a multiprotein complex containing the corepressor NCoR, histone deacetylase 3 (HDAC3), and a WD40-repeat protein TBL1. Surprisingly, the OHT-complexed ERalpha also recruited a chromatin-remodeling NuRD complex in which histone deacetylase 1 (HDAC1) is associated with several polypeptides including metastasis-associated protein 1/2 (MTA1/2), and SWI2/SNF2-related ATPase Mi2. Kinetic studies revealed that following OHT addition the recruitment of these HDAC complexes to pS2 or the c-myc promoter occurs in a sequential manner; the NCoR-HDAC3 complex is recruited earlier than the NuRD complex. Serial ChIP experiments indicated that the ER-NCoR-HDAC3 and ER-NuRD complexes are distinct, and they do not occupy the target gene promoter simultaneously. We also established a close temporal link between the appearance of the HDAC complexes at the E-responsive regions of pS2 and c-myc promoters, local hypoacetylation of specific lysine residues in N-terminal tails of histones H3 and H4, and disappearance of RNA polymerase II from the target gene loci. Collectively, our studies indicated that transcriptional repression by tamoxifen-bound ER at E-regulated gene promoters involves a dynamic interplay of multiple distinct chromatin-modifying/remodeling complexes.

Cell Line, Tumor↗

Promoter identity shapes splicing outcomes and fidelity.

Gene expression is a complex process subject to regulation at multiple functionally interconnected levels. One prominent example is the crosstalk between transcription and splicing regulation. Past work has shown that transcription can influence splicing in multiple ways, but a systematic investigation of this complex interplay is lacking. Here we employ massively parallel reporter assays of large combinatorial promoter-splice site libraries to dissect how promoter identity and transcription dynamics affect alternative splicing in human cells. We find that promoter identity, rather than expression level, exerts strong and highly context-specific effects on cassette exon inclusion, exceeding the effect of pharmacological inhibitors of transcription initiation or elongation. Groups of exons display coordinated promoter-dependent splicing behavior, and we identified predictive sequence and structural features underlying this sensitivity. Promoter and gene architecture also shape isoform diversity by modulating cryptic splice site usage. These findings present promoters as central regulators of splicing outcomes and fidelity.

Humans↗

Transcriptional regulation of cyclooxygenase 2 by bradykinin and interleukin-1beta in human airway smooth muscle cells: involvement of different promoter elements, transcription factors, and histone h4 acetylation.

Bradykinin and interleukin-1beta (IL-1beta) induce cyclooxygenase 2 (COX-2) in human airway smooth muscle cells. Here we extended our study to explore the gene transcriptional regulation. By transfection with various COX-2 promoter reporter constructs, we found that the bp -327-to-+59 promoter region was essential for COX-2 gene transcription by bradykinin and IL-1beta and that the cyclic AMP response element (CRE) was critical in bradykinin-induced transcription, whereas nuclear factor IL-6 and CRE and, to a lesser extent, nuclear factor-kappaB (NF-kappaB) were involved in IL-1beta-induced transcription. An electrophoretic mobility shift assay revealed that both bradykinin and IL-1beta elicited CRE-binding protein-1 (CREB-1) binding, and IL-1beta also elicited CCAAT/enhancer-binding protein beta and NF-kappaB binding to their respective elements in the COX-2 promoter. These transcription factors were associated with the COX-2 promoter, which was dynamically linked to different patterns of histone H4 acetylation by bradykinin and IL-1beta, as demonstrated by chromatin immunoprecipitation. We also revealed that endogenous prostaglandin E(2) was critical in bradykinin-induced COX-2 transcription initiation and involved in IL-1beta-induced COX-2 transcription at a latter stage. Our result provide the first evidence that COX-2 transcriptional regulation by different stimuli involves different promoter elements and transcription factors and is associated with chromatin remodeling after selective histone H4 acetylation in a stimulus-specific manner.

Acetylation↗

Viral replication through phase separation: Cytosolic and nuclear condensates.

Replication of many RNA and DNA viruses occurs within specialized intracellular hubs organized as membraneless biomolecular condensates (BCs) driven by liquid-liquid phase separation. As obligate intracellular parasites, viruses depend on the host cell machinery to complete their replication cycles and therefore actively remodel the intracellular environment to favor viral genome replication, transcription, and assembly. Cytosolic and nuclear phase-separated replication compartments (RC) provide concentrated and dynamic platforms that promote efficient interactions between viral genomes and viral or host proteins essential for infection. The formation of viral replication BCs is typically facilitated by viral proteins enriched in intrinsically disordered regions and low-complexity domains, which enable multivalent interactions with viral nucleic acids and cellular factors. These interactions are mediated by diverse biophysical forces, including hydrophobic and π interactions, hydrogen bonding, molecular crowding, and osmotic effects. Throughout infection, viral BCs remain highly dynamic, allowing continuous exchange of components and functional maturation of replication hubs. Their properties and activities are further regulated by post-translational modifications of viral and host proteins, such as phosphorylation, acetylation, and methylation. In this review, we summarize current evidence supporting liquid-liquid phase separation as a central organizing principle of viral RCs. We focus on representative RNA and DNA viruses that replicate in the cytosol or nucleus, highlighting virus-specific strategies, conserved mechanisms, and the consequences of BC formation for viral replication efficiency, host antiviral responses, and therapeutic intervention.

Phase Separation↗

Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

Animals↗

[Response to dynamic strain in human periosteal cells grown in vitro].

The objective of this program is to investigate the biological effect of dynamic strain on human periosteal cells in vitro. Using a well-established model, the Flexercell unit, we placed mechanical stress (50,000 microstrain, 1 Hz and sine wave) on human periosteal cells grown in collagen coated flexible membrane. The time points of proliferative and differentiative properties were assessed by means of cell counting, thymidine incorporation, synthesis of alkaline phosphatase and osteocalcin, and long term of mechanical load induced calcium nodules formation was also demonstrated. The results showed that the application of highly controlled strains exerted a significant effect on human periosteal cells by up regulation of osteogenic properties rather than exercised an influence on proliferation. The results suggested that the promoting effects of dynamic strain on human periosteal cells probably contribute to the biological function of mechanical loading bearing.

Biomechanical Phenomena↗

Radical approaches to probe protein structure, folding, and interactions by mass spectrometry.

This review describes mass spectrometry-based strategies and investigations to determine protein structure, folding dynamics, and protein-protein interactions in solution through the use of radical reagents. The radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources, and react with proteins on time scales that are less than those often attributed to structural reorganization and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt dynamic structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin and the binding of gelsolin to actin are among the systems presented to illustrate the approach.

Animals↗

Role of repolarization restitution in the development of coarse and fine atrial fibrillation in the isolated canine right atria.

INTRODUCTION: Although the role of action potential duration restitution (APD-R) in the initiation and maintenance of ventricular fibrillation (VF) has been the subject of numerous investigations, its role in the generation of atrial fibrillation (AF) is less well studied. The cellular and ionic basis for coarse versus fine AF is not well delineated. METHODS AND RESULTS: We measured APD-R during acetylcholine-mediated AF as well as during pacing (standard and dynamic protocols) in crista teriminalis, pectinate muscle, superior vena cava, and appendage of isolated canine arterially perfused right atria (n = 15). Transmembrane action potential (TAP), pseudo-ECG, and isometric tension development were simultaneously recorded. Acetylcholine flattened APD-R measured by both standard and dynamic protocols, but promoted induction of AF. AF was initially coarse, converting to fine within 3-15 minutes of AF. Coarse, but not fine AF was associated with dramatic fluctuations in tension development, reflecting wide variations in intracellular calcium activity ([Ca(2+)](i)). During coarse AF, APD-R data displayed a cloud-like distribution pattern, with a wide range of maximum APD-R slope (from 1.21 to 0.35). A maximum APD-R slope >1 was observed only in crista terminalis (3/10). The APD-R relationship was relatively linear and flat during fine AF. Reduction of [Ca(2+)](i) was associated with fine AF whereas augmentation of [Ca(2+)](i) with coarse AF. CONCLUSIONS: Our data indicate that while APD-R may have a limited role in the maintenance of coarse AF, it is unlikely to contribute to the maintenance of fine AF and that [Ca(2+)](i) dynamics determine the degree to which AF is coarse or fine.

Acetylcholine↗

Transcriptional regulation of VCAM-1 expression by tumor necrosis factor-alpha in human tracheal smooth muscle cells: involvement of MAPKs, NF-kappaB, p300, and histone acetylation.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the expression of adhesion molecules in airway resident cells and contribute to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB in TNF-alpha-induced expression of vascular cell adhesion molecule (VCAM)-1 were investigated in human tracheal smooth muscle cells (HTSMCs). TNF-alpha-enhanced expression of VCAM-1 protein and mRNA as well as phosphorylation of p42/p44 MAPK, p38, and JNK were significantly attenuated by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). Transfection with dominant negative mutants of MEK1/2, ERK1, ERK2, p38, and JNK attenuated TNF-alpha-induced VCAM-1 expression. Furthermore, TNF-alpha-induced VCAM-1 expression was significantly blocked by a selective NF-kappaB inhibitor helenalin. TNF-alpha-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF-alpha in HTSMCs transfected with VCAM-1-Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Most surprisingly, VCAM-1 expression was also significantly blocked by a selective inhibitor of p300, curcumin. NF-kappaB transcription factor and p300 were associated with the VCAM-1 promoter, which was dynamically linked to histone H3 acetylation stimulated by TNF-alpha, as determined by chromatin immunoprecipitation assay. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells (PMNs) to monolayer of HTSMCs, which was blocked by helenalin, U0126, SB202190, or SP600125. These results suggest that in HTSMCs, activation of MAPK pathways, NF-kappaB, and p300 is essential for TNF-alpha-induced VCAM-1 expression.

Acetylation↗

Mechanisms of circulatory homeostasis and response in Aplysia.

This review concerns the organization and function of arterial vasculature in Aplysia californica, especially the vasomotor reflexes that support circulatory homeostasis, and fixed patterns of response that may reroute blood flow during changes in behavioral state. The observations presented here raise three hypotheses for further study: 1) Arterial vasculature is functionally organized with precisely structured, independently regulated subdivisions; these are most evident for arterial systems serving digestive and reproductive processes; 2) arterial musculature is inherently responsive to local pressure changes, having both static and dynamic reflexes that promote efficient, evenly-distributed flow of blood; and 3) complex, long-lasting behaviors like egg laying have, as part of their makeup, equally prolonged and stereotypical changes in the pattern of circulation. Taken together, these observations support the view that maintenance and adjustment of blood flow in gastropod molluscs is an unexpectedly complex and highly integrated component of behavior.

Animals↗

Does resource availability, resource heterogeneity or species turnover mediate changes in plant species richness in grazed grasslands?

Grazing by large ungulates often increases plant species richness in grasslands of moderate to high productivity. In a mesic North American grassland with and without the presence of bison ( Bos bison), a native ungulate grazer, three non-exclusive hypotheses for increased plant species richness in grazed grasslands were evaluated: (1) bison grazing enhances levels of resource (light and N) availability, enabling species that depend on higher resource availability to co-occur; (2) spatial heterogeneity in resource availability is enhanced by bison, enabling coexistence of a greater number of plant species; (3) increased species turnover (i.e. increased species colonization and establishment) in grazed grassland is associated with enhanced plant species richness. We measured availability and spatial heterogeneity in light, water and N, and calculated species turnover from long-term data in grazed and ungrazed sites in a North American tallgrass prairie. Both regression and path analyses were performed to evaluate the potential of the three hypothesized mechanisms to explain observed patterns of plant species richness under field conditions. Experimental grazing by bison increased plant species richness by 25% over an 8-year period. Neither heterogeneity nor absolute levels of soil water or available N were related to patterns of species richness in grazed and ungrazed sites. However, high spatial heterogeneity in light and higher rates of species turnover were both strongly related to increases in plant species richness in grazed areas. This suggests that creation of a mosaic of patches with high and low biomass (the primary determinant of light availability in mesic grasslands) and promotion of a dynamic species pool are the most important mechanisms by which grazers affect species richness in high productivity grasslands.

Animals↗

Sequential histologic analysis of the myocardium after dynamic cardiomyoplasty: a study based on right ventricular endomyocardial biopsies.

Although dynamic cardiomyoplasty may promote clinical amelioration and mild improvement in left ventricular function in selected patients, whether cardiac reverse remodeling occurs after the procedure is not clear. We did not find histologic differences among right ventricular endomyocardial biopsy specimens taken before and after the surgery at 3 time periods. This result suggests that the procedure has no effect on the microscopic structure of the right ventricular myocardium. However, because reverse remodeling does not necessarily occur concomitantly in both ventricles, our conclusion cannot be extrapolated to the left ventricular chamber.

Adult↗

Molecular phylogeny and biogeography of the dung beetle genus Temnoplectron Westwood (Scarabaeidae: Scarabaeinae) from Australia's wet tropics.

The landscape of the Australian Wet Tropics can be described as "islands" of montane rainforest surrounded by warmer or more xeric habitats. Historical glaciation cycles have caused expansion and contraction of these rainforest "islands" leading to consistent patterns of genetic divergence within species of vertebrates. To explore whether this dynamic history has promoted speciation in endemic and diverse groups of insects, we used a combination of mtDNA sequencing and morphological characters to estimate relationships and the tempo of divergence among Australian representatives of the dung beetle genus Temnoplectron. This phylogenetic hypothesis shares a number of well-supported clades with a previously published phylogenetic hypothesis based on morphological data, though statistical support for several nodes is weak. Sister species relationships well-supported in both tree topologies, and a tree obtained by combining the two data sets, suggest that speciation has mostly been allopatric. We identify a number of speciation barriers, which coincide with phylogeographic breaks found in vertebrate species. Large sequence divergences between species emphasize that speciation events are ancient (pre-Pleistocene). The flightless, rainforest species appear to have speciated rapidly, but also in the distant past.

Animals↗

Shared governance in an integrated health care network.

The nursing practice model of shared governance is a key strategy for enhancing work environments in Magnet hospitals. Shared governance is a dynamic process that promotes collaboration, shared decision making, and accountability for practice through workforce empowerment. Although the principles of shared governance are universal, structure and process generally follow the needs of an organization based on its core values, mission, vision, and philosophy. This article details the shared governance structure of one health care network.

Adult↗

[Ethical foundations of institutional psychotherapy].

HISTORICAL ASPECTS: The idea behind this work is to have an ethical examination of the institutional psychotherapy movement which has long influenced French public psychiatry and which has progressively, since the 80s, been subject to growing doubts. In the first part, institutional psychotherapy is presented. It is a model for theoretical development and practice in psychiatric care. It came into being just following the end of the Second World War at the same time as modern medical ethics. Its principles come on the one hand, from recognition of asylums' pathogenic effects--which led to the crushing of the patient's being--and on the other, through recognition of the uniqueness of each person and the subjectivity of mental suffering. These elements gave rise to creativity within the world of medicine and, in the sector, generated the science of psychiatry which advocated for continuity in care (both inpatient and outpatient) and preventive work directed at the population. This movement called for the use of the institution in its dynamic aspect which promotes exchanges and allows patients to situate or resituate themselves in historic and symbolic dimensions. It privileges a high level of transversality, maximum communication, favouring speaking out loud and responsibility. It requires a permanent analysis of the institutional counter transference (emotional reactions of the caregivers involved, their interrelations and the social and material organization of the institution) which determines the therapeutic action itself. THEORICAL BASIS: In a second part, its theoretical foundations and its practice shall be investigated in light of the guiding concepts of medical ethics (justice, autonomy, beneficence, non-malfeasance). Institutional psychotherapy responds to the need for justice by considering the patient as a whole and by conceiving each patient as being like oneself despite the differences (associated with the mode of hospitalization, the social or diagnostic category). The principle of autonomy lies always at care's horizon, made concrete in practice by notions of habitability, orientation to place and time, references, by activities and by meetings. The principle of beneficence was the basis for round-the-clock intakes, the use of an established theoretical model and clinical practice centred on the patient's words. Institutional analysis attempts not to repeat the alienation felt by the patient, alienation being associated with the treatment environment, and draws from the principle of non-malfeasance. It therefore appears that the approach of institutional psychotherapy conforms with ethical requirements, given its major interest in the subject who aspires to find meaning in life. CURRENT SITUATION: The third part discusses current orientations in care related to the explosion in neuroscience and technology, the promotion of the citizen as an individual, and legal doctrine, budgetary constraints, and new demands made on psychiatry from the social and political domains. The widespread trend towards simplification, swollen with hypotheses from neuroscientific research, is progressively reducing mental illness to target symptoms. The recovery of the notion of citizenship through technological capture and ideological strains in contemporary culture have also affected a suspension of the subject as a thinking and desiring being, and exempted caregivers from considering transferral phenomena, indicated with the appearance of new signifiers: user, stress, plague, network. The new medical-technical jargon of scales, tables, and management participates in the same process of patient objectification and care compartmentalization. In this context, under the cover of science and generally good actions, psychiatry has become biologisized, whilst being diluted from its social aspect, even as it becomes more repressive for patients. CONCLUSION: This observation leads to the conclusion, in the fourth part, that there is a need for psychiatry, which within its own discipline has a hard time finding sufficient resources, to refocus itself ethically. This enlightenment could come within a multi-disciplinary ethical space, uniting practitioners and psychiatric caregivers, psychoanalysts, philosophers, sociologists, neurobiologists etc. The research should concern the legitimacy of the principles that underlie psychiatric action in its care and preventive aspects.

Ethics, Clinical↗

Hydride transfer in liver alcohol dehydrogenase: quantum dynamics, kinetic isotope effects, and role of enzyme motion.

The quantum dynamics of the hydride transfer reaction catalyzed by liver alcohol dehydrogenase (LADH) are studied with real-time dynamical simulations including the motion of the entire solvated enzyme. The electronic quantum effects are incorporated with an empirical valence bond potential, and the nuclear quantum effects of the transferring hydrogen are incorporated with a mixed quantum/classical molecular dynamics method in which the transferring hydrogen nucleus is represented by a three-dimensional vibrational wave function. The equilibrium transition state theory rate constants are determined from the adiabatic quantum free energy profiles, which include the free energy of the zero point motion for the transferring nucleus. The nonequilibrium dynamical effects are determined by calculating the transmission coefficients with a reactive flux scheme based on real-time molecular dynamics with quantum transitions (MDQT) surface hopping trajectories. The values of nearly unity for these transmission coefficients imply that nonequilibrium dynamical effects such as barrier recrossings are not dominant for this reaction. The calculated deuterium and tritium kinetic isotope effects for the overall rate agree with experimental results. These simulations elucidate the fundamental nature of the nuclear quantum effects and provide evidence of hydrogen tunneling in the direction along the donor-acceptor axis. An analysis of the geometrical parameters during the equilibrium and nonequilibrium simulations provides insight into the relation between specific enzyme motions and enzyme activity. The donor-acceptor distance, the catalytic zinc-substrate oxygen distance, and the coenzyme (NAD(+)/NADH) ring angles are found to strongly impact the activation free energy barrier, while the donor-acceptor distance and one of the coenzyme ring angles are found to be correlated to the degree of barrier recrossing. The distance between VAL-203 and the reactive center is found to significantly impact the activation free energy but not the degree of barrier recrossing. This result indicates that the experimentally observed effect of mutating VAL-203 on the enzyme activity is due to the alteration of the equilibrium free energy difference between the transition state and the reactant rather than nonequilibrium dynamical factors. The promoting motion of VAL-203 is characterized in terms of steric interactions involving THR-178 and the coenzyme.

Alcohol Dehydrogenase↗