Pedagogical view of model metabolic cycles.

The main purpose of this study was to present a simplified view of model metabolic cycles. Although the models have been elaborated with the Mathematica Program, and using a system of differential equations, the main conclusions were presented in a rather intuitive way, easily understandable by students of general courses of Biochemistry, and without any need of mathematical support. A change in any kinetic constant (Km or Vmax) of only one enzyme affected the metabolic profile of all the substrates of the cycle. In addition, it is shown how an increase in the Km or a decrease in the Vmax values of any particular enzyme promoted an increase of its substrate; the contrary occurred decreasing the Km or increasing the Vmax values.

Mathematica program: its use to simulate metabolic irreversible pathways and inhibition of the first enzyme of a pathway by its end product as visualized with the reservoir model.

The main objective of this report is to show the usefulness and versatility of the Mathematica program to simulate enzyme linear pathways and to depict the effect of changing the Vmax and/or Km values of one or more enzymes on the course of the reaction. In addition, analysis of the different types of inhibition of the first enzyme of the pathway by its end product is viewed with the reservoir model for enzyme kinetics. All the data shown here are quantitatively related to the kinetic constants of the implicated enzymes. Particular attention has been paid to calculate the time needed to achieve half of the possible total synthesis of the final product of a metabolic pathway.

Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase.

UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH(2)p) yielding uridine 5'-(ß,γ-methylenetriphosphate) (UppCH(2)p). The following bisphosphonates were not acceptors of UMP: alendronate, pamidronate, clodronate and etidronate. UDP-glucose serves as uridylyl donor to triphosphate derivatives of the mevalonate pathway: farnesyl (far-PPP), geranyl (ger-PPP) and isopentenyl (iso-PPP), with formation of farnesyl-tetraphosphouridine (far-ppppU); geranyl-tetraphosphouridine (ger-ppppU) and isopentenyl-tetraphosphouridine (iso-ppppU). The K(m) (mM) and V(max) (mU/mg protein) values determined for these substrates were: 0.32 ± 0.07 and 4.9 ± 0.6; 0.21 ± 0.06 and 5.7 ± 0.8; 0.51 ± 0.14 and 2.0 ± 0.2, respectively. The K(m) and V(max) values for methylenebisphosphonate were 1.1 ± 0.2 mM and 4055 ± 96 mU/mg protein, respectively.

Prediction of mortality in patients with major burns: clinical and biochemical factors.

OBJECTIVE: The plasma electric charge, in addition to clinical factors, was considered to improve the prediction of mortality in patients with major burns. METHODS: A software called PICAL 5.0 was used to determinate the plasma electric charge in 143 patients with major burns from the intensive care burn unit-Unfallkrankenhaus Berlin (Germany). In addition, a retrospective study with these patients was developed involving: (1) biochemical variables in the first 48 hours: pH value, [albumin], [Ca(2+)], etc; (2) clinical aspects: age, total body surface area and full-thickness surface area burned, diagnosis of inhalation injury, etc. A mortality predictive equation was calculated from univariate and multivariate logistic regression analyses in a set of randomly chosen participants and applied to a validation set of 35 participants. RESULTS: The importance of each ion and protein for the equilibrium in the plasma charge is determinant. In statistical multivariate analysis, age, total body surface area burned, pH value, and [Mg(2+)] were independently associated with mortality. CONCLUSIONS: [Na(+)], [HCO(3)(-)] (bicarbonate), and [Cl(-)] are the ions contributing the most to the plasma charge equilibrium in patients with major burns; a loss of 50% of plasma proteins in the first 48 hours is equivalent to the loss of 1 mmol/L of HCO(3)(-). Moreover, the consideration of plasma biochemical parameters in the first 48 hours may improve the mortality predictive equation of mortality for burned victims.

Refolding and characterization of methionine adenosyltransferase from Euglena gracilis.

Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 µmol/min/mg and K(m) values of approximately 85 and 260 µM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed ß-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.

Determination of enzymatic activities and metabolites in microliter sample size.

A new generation of spectrophotometers able to measure a wide range of absorbance in microliter aliquots is currently used for the determination of DNA, RNA, and proteins. The object of this article is to show that these instruments could be easily adapted for routine evaluation of enzymes and metabolites in 1-2-microl volumes of biological samples.

Isoelectric point, electric charge, and nomenclature of the acid-base residues of proteins.

The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins.

Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.

Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

Bisphosphonates activate the 5-fluorouracil/uracil phosphoribosyltransferase activity present in Saccharomyces cerevisiae cell extracts: implications for tumor treatments.

Most of the effects described for bisphosphonates (pC(R1)(R2)p) are related, directly or indirectly with a pyrophosphate moiety. Bisphosphonates are (i) analogs of pyrophosphate in the synthesis of ATP derivatives (AppC(R1)(R2)p) catalyzed by ligases and (ii) inhibitors of enzymes of the mevalonate pathway with substrates containing a terminal pyrophosphate. Searching for the role of bisphosphonates on other reactions involving pyrophosphate, we explored their effect on a phosphoribosyltransferase activity, present in Saccharomyces cerevisiae cell extracts, using 5-fluorouracil or uracil as substrates. Unexpectedly, bisphosphonates increased the initial rate of synthesis of 5-FUMP (from 5-fluorouracil and phosphoribosylpyrophosphate): etidronate (2.8+/-0.3 times); pamidronate (2.6+/-0.4 times); alendronate (2.5+/-0.6 times) and clodronate (2.0+/-0.1 times). Similar values for the synthesis of UMP (from uracil and phosphoribosylpyrophosphate) were obtained in the presence of bisphosphonates. The values of the activation constants determined for alendronate and clodronate for the synthesis of UMP were 0.05+/-0.02 mM and 0.32+/-0.22 mM, respectively. These results raise the possibility that bisphosphonates enhance the effect of 5-fluorouracil (or other uracil prodrugs) in the treatment of bone tumors or bone tumor metastases.

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

Improved application of the oscillating method for the isoelectric point determination of protein: potential connection with protein data banks.

The oscillating method (OM) for the theoretical determination of the pI values, one by one, of proteins and other macromolecules has been previously published [Sillero and Maldonado, Comput. Biol. Med 36 (2006) 157-166]. An improved application of the method, here named as improved oscillating method (IOM), allows the pI determination of group of proteins. This characteristic may be useful to explore the pI value and electric charge of family of enzymes. As an example the pI values of 1630 enzymes collected in a Swiss-Prot data bank (www.expasy.org), as belonging to the enzymes ligases (EC 6. 2. 1. *) is presented. The method also permits the determination of the pI value of any group of proteins stored in data banks provided that they can be supplied to the program in a FASTA format. Free access to IOM can be reached at http://www.bq.uam.es/otros/pical3.zip.

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

Protein superfamily evolution and the last universal common ancestor (LUCA).

By exploiting three-dimensional structure comparison, which is more sensitive than conventional sequence-based methods for detecting remote homology, we have identified a set of 140 ancestral protein domains using very restrictive criteria to minimize the potential error introduced by horizontal gene transfer. These domains are highly likely to have been present in the Last Universal Common Ancestor (LUCA) based on their universality in almost all of 114 completed prokaryotic (Bacteria and Archaea) and eukaryotic genomes. Functional analysis of these ancestral domains reveals a genetically complex LUCA with practically all the essential functional systems present in extant organisms, supporting the theory that life achieved its modern cellular status much before the main kingdom separation (Doolittle 2000). In addition, we have calculated different estimations of the genetic and functional versatility of all the superfamilies and functional groups in the prokaryote subsample. These estimations reveal that some ancestral superfamilies have been more versatile than others during evolution allowing more genetic and functional variation. Furthermore, the differences in genetic versatility between protein families are more attributable to their functional nature rather than the time that they have been evolving. These differences in tolerance to mutation suggest that some protein families have eroded their phylogenetic signal faster than others, hiding in many cases, their ancestral origin and suggesting that the calculation of 140 ancestral domains is probably an underestimate.

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase.

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'''-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.

Exploiting protein structure data to explore the evolution of protein function and biological complexity.

New directions in biology are being driven by the complete sequencing of genomes, which has given us the protein repertoires of diverse organisms from all kingdoms of life. In tandem with this accumulation of sequence data, worldwide structural genomics initiatives, advanced by the development of improved technologies in X-ray crystallography and NMR, are expanding our knowledge of structural families and increasing our fold libraries. Methods for detecting remote sequence similarities have also been made more sensitive and this means that we can map domains from these structural families onto genome sequences to understand how these families are distributed throughout the genomes and reveal how they might influence the functional repertoires and biological complexities of the organisms. We have used robust protocols to assign sequences from completed genomes to domain structures in the CATH database, allowing up to 60% of domain sequences in these genomes, depending on the organism, to be assigned to a domain family of known structure. Analysis of the distribution of these families throughout bacterial genomes identified more than 300 universal families, some of which had expanded significantly in proportion to genome size. These highly expanded families are primarily involved in metabolism and regulation and appear to make major contributions to the functional repertoire and complexity of bacterial organisms. When comparisons are made across all kingdoms of life, we find a smaller set of universal domain families (approx. 140), of which families involved in protein biosynthesis are the largest conserved component. Analysis of the behaviour of other families reveals that some (e.g. those involved in metabolism, regulation) have remained highly innovative during evolution, making it harder to trace their evolutionary ancestry. Structural analyses of metabolic families provide some insights into the mechanisms of functional innovation, which include changes in domain partnerships and significant structural embellishments leading to modulation of active sites and protein interactions.

Isoelectric point determination of proteins and other macromolecules: oscillating method.

A program written in Visual Basic has been developed to calculate the isoelectric point of proteins and other macromolecules bearing acid-basic residues. The pI value can be theoretically calculated with the precision required. The computer automatically supplies a representation of the charge of the protein versus pH values. The corresponding values can also be obtained, on command, in the form of table.

Pentose phosphate and calvin cycles: Similarities and three-dimensional views*.

The main object of this work is to present simplified and three-dimensional views of the pentose phosphate and Calvin cycles, emphasizing their functional and chemical similarities.

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1.

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k(cat) = 0.29+/-0.05 s(-1)) the transfer of AMP from the E-AMP-ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra- or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.

Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells.

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.

Influence of chronological aging on the survival and nucleotide content of Saccharomyces cerevisiae cells grown in different conditions: occurrence of a high concentration of UDP-N-acetylglucosamine in stationary cells grown in 2% glucose.

Saccharomyces cerevisiae cells (strain W303) grown in a minimal medium (containing 2% or 0.1% glucose) until exponential or stationary phase, were subjected to chronological aging in water, and yeast viability and nucleotide content were analyzed along several days of nutrient starvation. Cells collected in exponential phase (whether grown in the presence of 0.1% or 2% glucose) were viable up to five days and thereafter the viability decreased linearly with a half-survival rate of around eight days. ATP and other nucleoside triphosphates decreased similarly in both cases. Cells collected in stationary phase, and transferred to water, behaved differently whether grown in 0.1% or in 2% glucose, with a half-survival life of around nine and 28 days respectively. A double mutant in glycogen synthase (gsy1delta gsy2delta) and its isogenic wild-type strain, grown to stationary phase in 2% glucose, presented a similar half-survival life of around eight days. The W303 cells grown to stationary phase in the presence of 2% glucose showed a 7-fold increase of UDP-N-acetylglucosamine (UDP-GlcNAc) as compared with the level present in the cells grown in any of the other three metabolic situations. The nature of UDP-GlcNAc was established by MALDI-TOF ionization analysis. It is also worth noting that the rate of decay of NAD+ was lower than that of ATP in any of the situations here considered.