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I H Segel

Publications and source records attributed to I H Segel.

At least 37 records · Page 2Linked to original sources
PubMed1989

Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase.

At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no "substrate channeling" of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [35S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a "3'-phosphoadenosine 5'-phosphosulfate synthetase" complex.

Adenosine Monophosphate↗
PubMed1989

Rat liver ATP-sulfurylase: purification, kinetic characterization, and interaction with arsenate, selenate, phosphate, and other inorganic oxyanions.

ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 +/- 12 kDa and appears to be composed of identical 62 +/- 6-kDa subunits. At 30 degrees C and pH 8.0 (50 mM Tris-Cl buffer containing 5 mM excess Mg2+), the best preparations have "forward reaction" specific activities of about 20 and 2 units X mg protein-1 with MoO4(2-) and SO4(2-), respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, Kia = 0.87 mM, KmB = 0.18 mM, KmQ = 0.65 microM, Kiq = 0.11 microM, and KmP = 5.0 microM where A = MgATP, B = SO4(2-), Q = APS, and P = total PPi at 5 mM Mg2+. PPi is a mixed-type inhibitor with respect to MgATP and SO4(2-). SeO4(2-) is an alternative inorganic substrate with a Vmax about 20% that of SO4(2-). The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO4(2-) and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 +/- 0.5 X 10(-3) sec-1 (t1/2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of Pi, the MgATP reciprocal plot of the SO4(2-)-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO4(2-) and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO3- greater than ClO4- greater than FSO3- greater than NO3-. Thiosulfate is also competitive with SO4(2-) but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO4(2-), WO4(2-), CrO4(2-), and HAsO4(2-] promote the enzyme-catalyzed cleavage of MgATP to AMP and MgPPi. The ratio Vmaxf/KmA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of MgPPi before APS.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Phosphosulfate↗
PubMed1988

The general modifier ("allosteric") unireactant enzyme mechanism: redundant conditions for reduction of the steady state velocity equation to one that is first degree in substrate and effector.

The general unireactant modifier mechanism in the absence of product can be described by the following linked reactions: E + S k1 in equilibrium k-1 ES k3----E + P; E + I k5 in equilibrium k-5 EI; EI + S k2 in equilibrium k-2 ESI k4----EI + P; and ES + I k6 in equilibrium k-6 ESI where S is a substrate and I is an effector. A full steady state treatment yields a velocity equation that is second degree in both [S] and [I]. Two different conditions (or assumptions) permit reduction of the velocity equation to one that is first degree in [S] and [I]. These are (a) that k-2k3 = k-1k4 (Frieden, C., J. Biol. Chem. 239, pp. 3522-3531, (1964)) and (b) that the I-binding reactions are at equilibrium (Reinhart, G. D., Arch. Biochem. Biophys. 224, pp. 389-401 (1983)). It is shown that each condition gives rise to the other (i.e., if the I-binding reactions are at equilibrium, then k-2k3 must equal k-1k4 and vice-versa). If one assumes equilibrium for the I-binding steps, the velocity equation derived by the method of Cha (J. Biol. Chem. 243, pp. 820-825 (1968)) is apparently second degree in [I] (Segel, I. H., Enzyme Kinetics, p. 838, Wiley-Interscience (1975)), but reduces to a first degree equation when the relationship derived by Frieden is inserted. If one starts by assuming a single equilibrium condition for I binding, e.g., k-5[EI] = k5[E][I] or k-6[ESI] = k6[ES][I], then a traditional algebraic manipulation of the remaining steady state equations provides first degree expressions for the concentrations of all enzyme species and also discloses the Frieden relationship.

Binding Sites↗
PubMed1987

ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification.

ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.

Adenosine Triphosphate↗
PubMed1986

ATP sulfurylase-dependent assays for inorganic pyrophosphate: applications to determining the equilibrium constant and reverse direction kinetics of the pyrophosphatase reaction, magnesium binding to orthophosphate, and unknown concentrations of pyrophosphate.

A continuous, coupled, spectrophotometric assay is described in which the enzyme ATP sulfurylase is employed to measure the concentration of inorganic pyrophosphate (PPi) at equilibrium with known concentrations of inorganic orthophosphate (Pi) in the presence of excess inorganic pyrophosphatase (PPitase). In agreement with previous reports, the apparent equilibrium constant (Keq,app) of the PPi hydrolysis reaction was shown to decrease as the concentration of Mg2+ is increased. At pH 7.3, 30 degrees C, in the presence of 150 mM NaCl and 1 mM free Mg2+, Keq,app (calculated as [Pi]t2/[PPi]t) was 1950. Measurements of Keq,app at different total concentrations of Mg2+ and Pi permitted the determination of K0, the dissociation constant of the Mg-Pi complex. In 0.05 M Tris-Cl, pH 8.0, at 30 degrees C, K0 was 3.6 mM. In the presence of excess ATP sulfurylase, yeast PPitase catalyzed PPi formation from Pi with a specific activity (Vmax) of 9 units X mg protein-1 at pH 8.0, 30 degrees C, and 1 mM free Mg2+. Half-maximum reverse reaction velocity was observed at a total Pi concentration of 18 mM. (Under the same conditions, Vmax of the PPi hydrolysis reaction was 530 units X mg protein-1.) A radiochemical end point ("reaction-to-completion") assay for measuring unknown concentrations of PPi was devised. In the presence of excess 35S-adenosine-5'-phosphosulfate ([35S]APS) as the cosubstrate, 35SO2-4 formation was stoichiometric with added PPi. (The 35SO2-4 and [35S]APS are separated by adsorption of the latter onto charcoal.) The sensitivity of the assay can be adjusted by varying the specific radioactivity of the [35S]APS. In the absence of interfering substances, as little as 2 pmol of PPi per 1.0 ml assay volume can be measured. The sensitivity of the assay is reduced in the presence of ATP plus perchlorate (which synergistically inhibit the enzyme). However, if the bulk of the ATP is removed from perchloric acid extracts of tissues with glucose and hexokinase, initial intracellular levels as low as 1 microM can be measured. The possibility that most of the cellular PPi extracted with perchloric acid was originally enzyme bound is discussed.

Diphosphates↗
PubMed1985

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation.

Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP.

Adenosine Diphosphate↗
PubMed1985

Adenosinetriphosphate sulfurylase from Penicillium chrysogenum: steady-state kinetics of the forward and reverse reactions, alternative substrate kinetics, and equilibrium binding studies.

The kinetics of the forward ATP sulfurylase-catalyzed reaction were examined using a new assay based on 32PPi released from [gamma-32P]MgATP in the presence of inorganic sulfate. Replots yielded Vmaxf = 6.6 units mg protein-1, KmA = 0.13 mM, Kia = 0.33 mM, and KmB = 0.55 mM, where A = MgATP and B = SO2-4. Thiosulfate, a dead-end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio kcat/KmA was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO2-4, SeO2-4, MoO2-4, WO2-4, or CrO2-4. For SO2-4 and SeO2-4, the ratio was 5-6.5 X 10(4) M-1 S-1; for the others, the ratio was 5.8-7.3 X 10(5) M-1 S-1. The results support a random addition of MgATP and inorganic substrate. The kinetics of the reverse reaction were examined using a new assay based on 35SO2-4 release from [35S]APS (adenosine 5'-phosphosulfate) in the presence of MgPPi. Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded Vmaxr = 50 units mg protein-1, KmQ = 0.3 microM, Kiq = 0.04 microM, and KmP = 4 microM, where Q = APS and P = PPi (total of all species). MgATP and SO2-4 were both competitive with APS and noncompetitive with respect to MgPPi. Taken together with earlier results suggesting that APS is competitive with both MgATP and SO2-4 and that MgPPi is noncompetitive with respect to both substrates, the qualitative results point to a random A-B, ordered P-Q kinetic mechanism. The Scatchard plot for [35S]APS binding was curved, indicating either negative cooperativity or more than a single class of sites. [gamma-32P]MgATP displayed half-site saturation in the presence of saturating FSO-3.

Adenosine Triphosphate↗
PubMed1985

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation.

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.

Adenosine Triphosphate↗
PubMed1985

Purification and characterization of bile salt sulfotransferase from human liver.

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.

Adenosine Diphosphate↗
PubMed1985

Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum.

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10(5) greater than that remaining in the medium. The apparent Keq is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external 35SO42- exchanges with internal, unlabeled SO42- at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1 is characterized by the rapid release of 35SO42- when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t 1/2, 0.38 min). 35SO42- in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of 35SO42- induced by azide at pH 8.4 or unlabeled sulfate (t 1/2, 32 to 49 min). Early in the 35SO42- accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated 35SO42-. The kinetics of 35SO42- accumulation is consistent with the following sequential process: medium----pool 1----pool 2. Monensin (33 microns) accelerates the transfer of 35SO42- from pool 1 to pool 2. Valinomycin (0.2 microM) and tetraphenylboron- (1 mM) retard the transfer of 35SO42- from pool 1 to pool 2. At the concentrations used, neither of the ionophores nor tetraphenylboron- affect total 35SO42- uptake. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.

Azides↗
PubMed1985

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.

Amino Acids↗
PubMed1984

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization.

Adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 59,000-60,000 and is composed of two 30,000-dalton subunits. At 30 degrees C, pH 8.0 (0.1 M Tris-chloride buffer), 5.5 microM APS, 5 mM MgATP, 5 mM excess MgCl2, and "high" salt (70-150 mM (NH4)2SO4), the most highly purified preparation has a specific activity of 24.7 units X mg of protein-1 in the physiological direction of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation of APS kinase. APS kinase is subject to potent substrate inhibition by APS. In the absence of added salt, the initial velocity at 5 mM MgATP plus 5 mM Mg2+ is maximal at about 1 microM APS and half-maximal at 0.2 and 4.4 microM APS. In the presence of 200 mM NaCl or 70-150 mM (NH4)2SO4, the optimum APS concentration shifts to 4-6 microM APS; the half-maximal values shift to 1-1.3 and 21-27 microM APS. The steady state kinetics of the reaction were investigated using a continuous spectrophotometric assay. The families of reciprocal plots in the range 0.25-5 mM MgATP and 0.8-5.1 microM APS are linear and intersect on the horizontal axis. Appropriate replots yield KmMgATP = 1.5 mM, KmAPS = 1.4 microM, and Vmax, = 38.7 units X mg of protein-1. Excess APS is an uncompetitive inhibitor with respect to MgATP (K1APS = 23 microM). PAPS, the product of the forward reaction, is also uncompetitive with MgATP. PAPS is not competitive with APS. In the reverse direction, the plots have the characteristics of a rapid equilibrium ordered sequence with MgADP adding before PAPS. The kinetic constants are KmPAPS = 8 microM, KiMgADP = 560 microM, and Vmaxr = 0.16 units X mg of protein-1. Iso-PAPS (the 2'-phosphate isomer of PAPS) is competitive with PAPS and uncompetitive with respect to MgADP (Ki = 6 microM). APS kinase is inactivated by phenylglyoxal, suggesting the involvement of an essential argininyl residue. MgATP or MgADP at 10 Ki protect against inactivation. APS or PAPS at 600 and 80 Km, respectively, are ineffective alone, but provide nearly complete protection in the presence of 0.1 Ki of MgADP or MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics↗
PubMed1983

Kinetics of enzymes subject to very strong product inhibition: analysis using simplified integrated rate equations and average velocities.

Enzymes which catalyze energetically unfavorable reactions in the physiological direction are likely to be strongly inhibited by the reaction products. (Some energetically favorable reactions may also display strong "product inhibition" when assayed in the reverse direction.) In some cases, the inhibition caused by an accumulating product is so potent that true initial velocities cannot be directly determined using conventional assay methods. Continuous removal of the inhibitory product may be mitigated against by the nature of the assay or the unavailability of the appropriate coupling enzyme. It can be shown that if (a) only one inhibitory product is allowed to accumulate and (b) the substrate concentrations remain essentially constant over the assay period (i.e. Kproduct less than or equal to 10(-2)Ksubstrate, so that the decreasing reaction rate stems only from progressive product inhibition), then plots of reciprocal average (apparent) velocity (i.e. 1/v = t/[P]) versus [P] are linear and extrapolate to 1/v0, the reciprocal of the initial uninhibited velocity at the fixed substrate concentrations. Intercept replots give the usual initial velocity reciprocal plot patterns and permit Vmax and the substrate Km's to be determined. Slope replots are diagnostic of the type of inhibition exerted by the accumulating product and permit the inhibition constants to be determined. If all the appropriate coupling enzymes are available, some kinetic mechanisms can be diagnosed using data derived from the reaction progress curves in the presence of one accumulating product at a time.

Enzyme Inhibitors↗
PubMed1983

ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism.

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.

Kinetics↗
PubMed1982

ATP sulfurylase from higher plants : purification and preliminary kinetics studies on the cabbage leaf enzyme.

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.

Journal Article↗