Initial Velocity Studies Research Articles

Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties.

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.

Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.

Kinetic mechanism of the reaction catalyzed by nuclear histone acetyltransferase from calf thymus.

The kinetic mechanism for calf thymus histone acetyltransferase A has been determined from the initial velocity studies. The kinetic patterns at low substrate concentrations suggest that the reaction proceeds via two half-reactions as in a ping-pong pathway with the formation of an acetyl-enzyme intermediate. Such acetyl-enzyme has been isolated and found to be chemically competent. In addition, product inhibition patterns by coenzyme A are consistent with a hybrid ping-pong mechanism. These findings indicate that the acetyltransferase A from calf thymus has two separate and independent binding sites, one for each of the two substrates. Consequently, the mechanism constructed for the acetyltransferase A catalyzed reaction may be described as a double-displacement, two-site ping-pong mechanism.

Kinetic studies on the reaction catalyzed by DNA ligase from calf thymus

Kinetic analysis of the reaction catalyzed by calf thymus DNA ligase (EC 6.5.1.1) has been carried out using [5′- 32P]nicked DNA as substrate. The results of initial velocity and product inhibition studies indicate that the ligase reaction is likely to proceed through the ‘uni-uni uni-bi ping-pong’ mechanism. The order of substrate addition and product release is as follows: ATP, PP i, nicked DNA, sealed DNA and 5′-AMP. The true K m values for ATP and for nicked DNA (5′-phosphoryl ends) were 2 μM and 0.11 μM, respectively. The turnover number was estimated to be 7 sealing events per min dATP was an inhibitor competitive with ATP ( K i = 25 μM). The addition of 0.5 mM spermine or 5 mM spermidine resulted in an increase in the apparent K m for nicked DNA as well as in the apparent V, whereas 0.1 KCl increased only the apparent K m for nicked DNA. Neither polyamine nor KCl affected the apparent K m for ATP. The ligase reaction was not significantly affected by aphidicolin and various phosphate compounds tested.

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies.

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.

Purification and properties of carnitine acetyltransferase from rat liver.

Carnitine acetyltransferase was purified from rat liver after induction of the enzyme by feeding with di(2-ethylhexyl)phthalate. Two enzyme sources were used: the mitochondrial fraction and the homogenate of the liver. The purification procedure was essentially the same for the two enzyme sources. The enzyme purified from the mitochondrial fraction consisted of two different polypeptides with molecular weights of 36,500 and 27,000, whereas that from the homogenate consisted of one polypeptide with a molecular weight of 67,500. Amino acid compositions and peptide maps of the limited proteolytic products of the two enzyme preparations were nearly the same. Their antibodies were cross-reactive. Catalytic properties of the two preparations were nearly the same: the specific enzyme activities, double reciprocal plots of initial velocity study, substrate specificities for acylcarnitines having various carbon chain lengths, apparent Michaelis constants for substrates. On electrophoresis of the immunoprecipitate obtained after incubation of the mitochondrial extract, the two immunoreactive polypeptides with molecular weights of 36,500 and 27,000 were found. But only one polypeptide, with molecular weight of 67,500, was detected when the protease inhibitors were added to the mitochondrial extract. It was concluded that the enzyme in the mitochondrial fraction was a monomeric form but was converted into a dimeric form by proteolytic modification after the disruption of mitochondria. The preparation from the post-mitochondrial fraction, which had a lower specific activity, contained two polypeptides whose molecular weights were 69,000 and 67,500. They could not be separated from each other throughout the purification. The peptide maps of the products of the limited proteolysis were very similar.

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit.

The kinetic mechanism for adenosine cyclic 3',5'-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg2+ to a second site. Increasing the Mg2+ results in a 5-6-fold decrease in V,,, in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/KMgATP in the absence of KCl and a 13-fold increase in V/KMdTP at 0.1 M KC1. The effect of increasing free Mg2+ on V,,, and V/K was also obtained with MgITP (20% the V,,, with MgATP) and MgGTP (10% the V,,, with MgATP) as substrates. The dissociation constant for Mg2+ from E-Ser-peptide-Mg2+ and central complexes is 2-3 mM. At low concentrations of free Adenosine cyclic 3',5'-monophosphate dependent protein kinase catalyzes the phorphorylation of a variety of proteins according to

Retinol dehydrogenase from bovine retinal rod outer segments. Kinetic mechanism of the solubilized enzyme.

Retinol dehydrogenase solubilized by Lubrol 12A9 from bovine retinal rod outer segments forms mixed micelles of Stokes radius 8.5 nm. The kinetic properties of the solubilized retinol dehydrogenase were examined and retinaldehyde reduction and retinol oxidation were seen to proceed at pH 8.3 by a sequential Ordered Bi Bi mechanism. This conclusion was supported by bisubstrate initial velocity studies, dead-end and product inhibition. The kinetic mechanism of retinol dehydrogenase is not altered by the effect of Lubrol until a concentration of 2 mM is reached, at which the detergent lowers the values of the Michaelis and dissociation constants. The catalytic rate of the retinol dehydrogenase is significantly lowered by detergent in the range of pH 3 to 9.

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase ( threo- d s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn 2+ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 m m. The Michaelis constant ( K m ) and dissociation constant ( K ib ) for isocitrate were 0.16 m m; and K m and dissociation constant ( K ia ) for NAD were 0.34 m m. The inhibition constant for NADH was 0.02 m m. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.

Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli.

The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects. The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF. At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme. The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply. However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4. The choice of conditions for analysis of progress-curve data is discussed in the Appendix.

Kinetic mechanism of threonyl-tRNA synthetase from human placenta.

Using purified enzyme and homologous tRNA, we have investigated the order of substrate binding and product release for the human placental threonyl-tRNA synthetase by isotope exchange, initial velocity, dead-end inhibition and product inhibition studies. The kinetic patterns obtained from these studies are consistent with a unique Bi Uni Uni Bi ping-pong mechanism. The order of addition of the first two substrates ATP and threonine is random, while the release of products follows an obligatory sequence, with AMP as the last product to dissociate from the enzyme.

Identification and kinetic studies of an inducible mannokinase from a Streptomyces strain

Crude extracts from cells of a Streptomyces strain isolated from a palm-grove soil and grown on different carbon sources showed a constitutive glucokinase. Specifically inducible kinase activity for mannose was found in cells grown on mannose or β- d-mannan. The activity on mannose was due to a highly specific mannokinase (ATP: d-mannose 6-phosphotransferase, EC 2.7.1.7) which has been separated from the glucokinase by Ultrogel AcA 54 gel filtration chromatography. Initial velocity and inhibition product studies were carried out to investigate the reaction pathway. In the absence of products, reciprocal plots intersecting on the abscissas were observed when either mannose or ATP concentration was varied in the presence of several fixed concentrations of the non-varied substrate. K m values for mannose and Mg-ATP complex are 0.33 and 1.1 mM, respectively. In the product inhibition studies, mannose-6- P was observed to be competitive with mannose and non-competitive with Mg-ATP. The reverse was observed when ADP was used as inhibitor. These data are consistent with a random sequential Bi-Bi mechanism with two dead-end ternary complexes.

ATP Sulfurylase from Higher Plants

ATP sulfurylase was purified extensively from green cabbage (Brassica capitata L.) leaf. The enzyme appears to be an asymmetric dimer composed of 57,000 dalton subunits. Initial velocity and product inhibition studies of the forward and reverse reactions point to an obligately ordered kinetic mechanism with MgATP adding before MoO(4) (2-) (or SO(4) (2-)). and MgPPi leaving before AMP + MoO(4) (2-) (or adenosine-5'-phosphosulfate [APS]). The addition of excess purified fungal APS kinase to assay mixtures increased the rate of (35)SO(4) (2-) incorporation and MgPPi formation and extended the linearity of the forward reaction. This effect can be ascribed to the continual removal of APS, a potent product inhibitor of ATP sulfurylase. The specific activities of the enzyme in the APS synthesis, molybdolysis, MgATP synthesis, and sulfate-dependent [(32)P]-MgPPi-MgATP exchange assays were 3.3, 38, 38, and 4.3 micromole product formed per minute per milligram protein, respectively.

Purification, characterization and inhibition by MK-401 of Fasciola hepatica phosphoglyceromutase

Phosphoglyceromutase (EC 2.7.5.3) of Fasciola hepatica was purified 1390-fold to homogeneity. The enzyme had a molecular weight of 120 000 and was a tetramer composed of identical 30 000 molecular weight subunits. The enzyme was 2,3-diphosphoglyceric acid dependent, possessed reactive sulfhydryl groups and was inhibited irreversibly by iodoacetamide, and N-ethylmaleimide and reversibly by p-chloromercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). Initial velocity studies suggest that reaction occurred via a sequential mechanism and that MK-401 was a competitive inhibitor versus both 3-phosphoglyceric acid and 2,3-diphosphoglyceric acid.

Purification and properties of succinyl-CoA:3-oxo-acid CoA-transferase from rat brain.

Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by G-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 mumol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate leads to acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 mM acetoacetate. Apparent Km values were 70 microM for acetoacetate and 156 microM for succinyl-CoA (the forward reaction), and 59 microM for acetoacetyl-CoA and 25 mM for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.

Kinetic analysis of dye-linked dehydrogenases and related enzymes where the electron acceptor binds twice during the catalytic sequence

Five possible kinetic mechanisms that are of relevance to the study of dye-linked dehydrogenases are examined in detail. It is shown that three of these mechanisms are linear and the remaining two are hyperbolic in double reciprocal space. These features allow three mechanisms to be distinguished by initial velocity studies alone, and product inhibition studies may sometimes but not always allow further resolution. A novel analysis of substrate inhibition is presented and finally, two relevant studies in the literature are discussed in the context of the present analysis.

Candida utilis NAD + kinase: Kinetic and inhibition studies with ADP

1. 1. Initial velocity and product inhibition studies using ADP were carried out on cytoplasmic NAD + kinase (ATP:NAD 2′ phosphotransferase, EC 2.7.1.23) purified from Candida utilis. Initial velocity studies were also carried out on a sample of chicken liver NAD +, kinase. 2. 2. The data indicate both enzymes followed a sequential mechanism of reactant binding. 3. 3. Product inhibition studies on C. utilis NAD + kinase suggest the mechanism of NAD +, ATP addition is best described as rapid equilibrium random with multiple binding of ADP to the free enzyme and the E·ATP and E·NAD + complexes. 4. 4. The characteristics of this enzyme, prepared from several sources, are briefly summarized.

Kinetic mechanism of beef pancreatic L-asparagine synthetase.

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.

Kinetic mechanism of assimilatory NADH:nitrate reductase from Chlorella.

Chlorella nitrate reductase catalyzes the reduction of nitrate to nitrite by NADH. Initial velocity studies showed that the kinetic mechanism is sequential, indicating that both substrates must bind to the enzyme before any products are released. Product inhibition with NAD and nitrite showed that competitive inhibition was observed when the inhibitor was similar to the varied substrate, while noncompetitive inhibition was observed when the inhibitor was dissimilar to the varied substrate. Likewise, dead-end inhibition with adenosine 5'-diphosphoribose and thiocyanate showed competitive inhibition when the inhibitor was similar to the varied substrate and noncompetitive inhibition when the inhibitor was dissimilar to the varied substrate. These results indicate that Chlorella nitrate reductase follows a random bi bi kinetic mechanism. Phosphate was found to stimulate NADH:nitrate reductase activity and 2-fold. The NADH:cytochrome c reductase activity associated with nitrate reductase was not affected by phosphate suggesting the effect of phosphate is on the nitrate-reducing moiety of the enzyme. Phosphate increases Vmax but has no effect on the apparent Km for nitrate.

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.