Biomedical subjects
Publications and source records attributed to R G Duggleby.
Acetolactate synthase catalyses the first step in branched-chain amino acid biosynthesis. The bacterial enzyme contains two large and two small subunits but there is only limited and circumstantial evidence for a small subunit in the eukaryotic enzyme. Here this evidence is summarised and protein sequences of two putative eukaryotic small subunits, from a yeast and a red alga, are presented.
Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.
MOTIVATION: The derivation of steady-state equations is frequently carried out in enzyme kinetic studies. Done manually, this becomes tedious and prone to human error. The computer programs now available which are able to accept reaction mechanisms of some complexity are focused only on the strict steady-state approach. RESULTS: Here we present a computer program called REFERASS, with a short computation time and a user-friendly format for the input and output files, able to derive the strict steady-state equations and/or those corresponding to the usual assumption that one ore more of the reversible steps are in rapid equilibrium. This program handles enzyme-catalysed reactions with mechanisms involving up to 255 enzyme species connected by up to 255 reaction steps, subject to limits imposed by the memory and disk space available.
UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3- bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.
Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439-445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenching is due to some perturbation of the local environment of Trp-487 rather than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence quenching upon addition of cofactors.
Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single concerned histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the Km for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding.
UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.-L. & Chang, H.-Y. (1993) FEBS Lett. 329, 153-158] and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration.
Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with 3H-labelled Ado and dAdo, samples were taken at 3 sec intervals and progress curves for the 3H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert to Ado predominantly to AMP, while dAdo is converted first to Hyp and the to AMP. The presence of dCf had little effect upon Ado metabolism by induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo-->dIno-->Hyp-->IMP-->sAMP-->AMP. dCf does not significantly affect the conversion of Ado-->AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.
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Product inhibition studies of reversible reactions can give unusual kinetic patterns. A plot of rate versus substrate concentration may appear to be sigmoidal and the double reciprocal plot will show upward curvature. This apparent positive cooperativity is due to the simultaneous occurrence of both the forward and reverse reaction. By fitting the appropriate rate equation to the data it is possible to determine the maximum velocity and Michaelis constant for both forward and reverse reaction from experiments conducted in one direction only.
The bifunctional enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase-IMP cyclohydrolase has been purified 780-fold to apparent homogeneity from human CCRF-CEM leukemia cells, completed with chromatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a Ks value for 5-formamidoimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 microM. The following purine nucleotide derivatives were potent competitive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosphate (Ki = 0.094 +/- 0.024 microM), xanthosine 5'-monophosphate (Ki = 0.12 +/- 0.01 microM), 2-fluoroadenine arabinoside 5'-monophosphate (Ki = 0.16 +/- 0.02 microM), 6-mercaptopurine riboside 5'-monophosphate (Ki = 0.20 +/- 0.02 microM), adenosine N1-oxide 5'-monophosphate (Ki = 0.28 +/- 0.03 microM), and N6-(carboxymethyl)adenosine 5'-monophosphate (Ki = 1.7 +/- 0.42 microM). The pH dependencies of Vmax and Vmax/Ks values for IMP cyclohydrolase are consistent with a single ionizable amino acid residue (pKa = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pKa = 7.57 +/- 0.14) which must be unprotonated for FAICAR to bind. The pKa values of 5.81 +/- 0.03 and 9.41 +/- 0.04 determined for FAICAR indicate that ionization of the substrate does not contribute significantly to the pH effects observed. Chemical modification of IMP cyclohydrolase provides evidence for arginine and cysteine residues at the active site, and roles for these residues in the mechanism of catalysis are proposed.
Protection against hypoxic injury by supraphysiological glycine and alanine concentrations was investigated in the isolated perfused rat kidney (IPRK). 23Na NMR detects consistent increases in total renal Na in IPRK during hypoxic perfusion. Increasing the concentration of glycine and alanine to 5 mM each produced a 34% (p < 0.001) reduction in the increase in total renal Na following 30 minutes of hypoxia compared to a matched control group supplemented with 5 mM each of serine and glutamine. There was also a trend (p = 0.067) to improvement in the fractional excretion of sodium (FENa) in the glycine plus alanine treated group. Hypoxic alterations of other physiological parameters were not prevented by supraphysiological glycine plus alanine. This suggests that monitoring total renal Na is a more sensitive method of defining renal injury and protection than monitoring changes in FENa, fractional excretion of potassium (FEK) and inulin clearance.
Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.
There are several advantages to the use of progress curves to analyze the the kinetic properties of enzymes but most studies still rely on rate measurements. One of the reasons for this may be that progress curve analysis relies on the enzyme and the reactants being completely stable under assay conditions. Here a method is described that relaxes this requirement and allows progress curve analysis to be applied to unstable enzymes. The procedure is based on a combination of numerical integration and non-linear regression to fit rate equations to the progress curve data. The analysis is verified using simulated data and illustrated by application to the reaction catalyzed by alkaline phosphatase, measured in the presence of 10 mM EGTA where it has a half-life of 3 1/2 min. The method may also be applied to other experimental systems where the development over time reveals important properties but where an analytical solution of the underlying model is not known. This extension is illustrated by two systems: the coupled reactions catalyzed by pyruvate kinase and lactate dehydrogenase under conditions where both enzymes have similar activity; and the transient-state kinetics of the reaction catalyzed by glutamate dehydrogenase.
The molecular weights of proteins or protein subunits are frequently estimated from the electrophoretic mobility in the presence of sodium dodecyl sulphate, or from the elution volume in gel filtration chromatography. Using a series of standards of known size, a calibration curve is prepared which often yields a straight line in a semilogarithmic plot; interpolation from this line then gives the size of the sample. While this method is simple to apply, it does not give any estimate of the accuracy of the determined size. Furthermore there is no objective criterion for deciding if the calibration curve is linear, or a procedure for dealing with non-linear calibration curves. Here I report a method and a computer program which uses appropriate statistical methods to select the best straight or curved calibration line. The size of the unknown is then interpolated and an estimate or the error in this value is calculated.
Hypoxic injury in the isolated perfused rat kidney (IPRK) was monitored using 23Na-NMR in the presence or absence of 1.5 and 15 mM dimethylthiourea (DMTU) or 15 mM dimethylsulphoxide (DMSO) before and after inducing hypoxia. Hypoxia induced a prompt exponential increase in total renal 23Na+, renal vascular resistance, and sodium excretion and decreased inulin clearance and adenine nucleotides and reduced glutathione concentrations. Lipid peroxide metabolites were unaltered. The increase in 23Na+ was significantly reduced (P < 0.001) by both DMTU and DMSO although hypoxic perturbations of function and biochemical parameters were not. Posthypoxic increases in renal 23Na+ include approximately 10% from the intratubular compartment, but principally reflect the intracellular and interstitial compartments. The results demonstrate that 23Na-NMR is a sensitive indicator of hypoxic renal injury in intact kidney and suggest that DMTU and DMSO protect against hypoxic injury by a mechanism independent of free radical-binding.
The binding and release of hormones and growth factors are often relatively slow processes under biological conditions. Consequently, a knowledge of the underlying rate constants may be of greater physiological relevance than the equilibrium constant. Here we show how, by following a single time course of binding, the rate constants for both binding and release can be determined. The ratio of these rate constants allows the binding constant to be calculated. A nonlinear regression computer program is described which facilitates these calculations and which provides estimates and standard errors of the constants determined. The method is illustrated by the binding of human growth hormone to the human growth hormone binding protein, and the binding of ovine prolactin to the rabbit prolactin receptor.