scholarly journals The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 139 (1) ◽  
pp. 27-35 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Exchange Reaction ◽  
Initial Rate ◽  
Leaf Tissue ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Sequential Mechanism ◽  
Spinach Leaf ◽  
Atp Sulphurylase

1. Sulphate-dependent PPi–ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP2− and MgP2O72−; ATP sulphurylase activity was not correlated with the concentration of free Mg2+. 2. Sulphate-dependent PPi–ATP exchange was independent of PPi concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PPi–ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [32P]ATP from [32P]PPi and adenosine 5′-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PPi and adenosine 5′-sulphatophosphate. 5. The synthesis of ATP from PPi and adenosine 5′-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PPi and adenosine 5′-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5′-sulphatophosphate and non-competitive with respect to PPi. It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP2− was the first product to react with the enzyme and MgP2O72− was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5′-sulphatophosphate could not be demonstrated.

scholarly journals Kinetic studies with the low-Km aldehyde reductase from ox brain

1985 ◽  
Vol 227 (2) ◽  
pp. 621-627 ◽  
Author(s):  
C M Ryle ◽  
K F Tipton
Keyword(s):  
Initial Velocity ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Binary Complex ◽  
Aldehyde Reductase ◽  
Apparent Dissociation Constant ◽  
Displacement Mechanism ◽  
Sequential Mechanism ◽  
Inhibition Studies

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

scholarly journals Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

1972 ◽  
Vol 127 (1) ◽  
pp. 237-247 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Substrate Specificity ◽  
Adenosine Triphosphate ◽  
Leaf Tissue ◽  
Coupled System ◽  
Thiol Group ◽  
Ph Optimum ◽  
Spinach Leaf ◽  
Inorganic Sulphur ◽  
Atp Sulphurylase

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [32P]PPi–ATP exchange. The enzyme was separated from Mg2+-requiring alkaline pyrophosphatase (which interferes with the PPi–ATP-exchange assay) and from other PPi–ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PPi–ATP exchange; Km (sulphate) was 3.1mm, Km (ATP) was 0.35mm and the pH optimum was 7.5–9.0. The enzyme was insensitive to thiol-group reagents and required either Mg2+ or Co2+ for activity. 3. The enzyme catalysed [32P]PPi–dATP exchange; Km (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [32P]PPi–ATP exchange and competed with sulphate; Km (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PPi–ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5′-[35S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg2+-dependent alkaline pyrophosphatase (also prepared from spinach) with [35S]sulphate and ATP as substrates; adenosine 5′-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.

scholarly journals Adenosine 5′-sulphatophosphate kinase activity in spinach leaf tissue

1973 ◽  
Vol 134 (2) ◽  
pp. 565-579 ◽  
Author(s):  
J. N. Burnell ◽  
J. W. Anderson
Keyword(s):  
Kinase Activity ◽  
Leaf Tissue ◽  
Nucleotidase Activity ◽  
Specific Enzyme ◽  
Ph Optimum ◽  
Spinach Leaf ◽  
Hydrolysis Of ◽  
Isolated Chloroplasts ◽  
Atp Sulphurylase

1. An F−-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PPi. The pH optimum of the enzyme was 7.5; Km (3′-AMP) was approx. 0.8mm and Km (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg2+-dependent pyrophosphatase, free from F−-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F−-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.

scholarly journals Estimation of the steady-state cyclic electron flux around PSI in spinach leaf discs in white light, CO2-enriched air and other varied conditions

2013 ◽  
Vol 40 (10) ◽  
pp. 1018 ◽  
Author(s):  
Jiancun Kou ◽  
Shunichi Takahashi ◽  
Riichi Oguchi ◽  
Da-Yong Fan ◽  
Murray R. Badger ◽  
...  
Keyword(s):  
Steady State ◽  
Electron Flux ◽  
Leaf Tissue ◽  
Linear Electron ◽  
Total Electron ◽  
Leaf Discs ◽  
Relative Water ◽  
Photochemical Yield ◽  
Spinach Leaf ◽  
Upper Estimate

Cyclic electron flux (CEF) around PSI is essential for efficient photosynthesis and aids photoprotection, especially in stressful conditions, but the difficulty in quantifying CEF is non-trivial. The total electron flux through PSI (ETR1) and the linear electron flux (LEFO2) through both photosystems in spinach leaf discs were estimated from the photochemical yield of PSI and the gross oxygen evolution rate, respectively, in CO2-enriched air. ΔFlux = ETR1 – LEFO2 is an upper estimate of CEF. Infiltration of leaf discs with 150 μM antimycin A did not affect LEFO2, but decreased ΔFlux 10-fold. ΔFlux was practically negligible below 350 μmol photons m−2 s−1, but increased linearly above it. The following results were obtained at 980 μmol photons m−2 s−1. ΔFlux increased 3-fold as the temperature increased from 5°C to 40°C. It did not decline at high temperature, even when LEFO2 decreased. ΔFlux increased by 80% as the relative water content of leaf discs decreased from 100 to 40%, when LEFO2 decreased 2-fold. The method of using ΔFlux as a non-intrusive upper estimate of steady-state CEF in leaf tissue appears reasonable when photorespiration is suppressed.

scholarly journals Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma

1994 ◽  
Vol 301 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S Lyle ◽  
D H Geller ◽  
K Ng ◽  
J Westley ◽  
N B Schwartz
Keyword(s):  
Steady State ◽  
Initial Velocity ◽  
Kinase Activity ◽  
Forward Direction ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Reverse Direction ◽  
Aps Kinase ◽  
Ordinate Axis ◽  
Atp Sulphurylase

ATP-sulphurylase catalyses the production of adenosine 5′-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3′-phosphate 5′-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.

scholarly journals Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes

1992 ◽  
Vol 287 (1) ◽  
pp. 145-150 ◽  
Author(s):  
G T M Henehan ◽  
K F Tipton
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Human Erythrocytes ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Sequential Mechanism ◽  
Kinetics Of

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear ‘mixed inhibition’ pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Properties of malate dehydrogenase isolated from Methanospirillum hungatii

1979 ◽  
Vol 25 (2) ◽  
pp. 192-200 ◽  
Author(s):  
G. D. Sprott ◽  
R. C. McKellar ◽  
K. M. Shaw ◽  
J. Giroux ◽  
W. G. Martin
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Reaction Mechanism ◽  
Malate Dehydrogenase ◽  
Initial Rate ◽  
Rate Data ◽  
Cell Extracts ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Variable Substrate

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, α-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by α-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. α-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to α-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of initial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was preferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells with respect to molecular weight (61 700) and reaction mechanism, but may be regulated differently.

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