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.     

Allosteric inhibition via R-state destabilization in ATP sulfurylase from Penicillium chrysogenum

The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), was determined to 2.6 A resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 A movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition by destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.     

Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum

The effects of temperature on the initial velocity kinetics of allosteric ATP sulfurylase from Penicillium chrysogenum were measured. The experiments were prompted by the structural similarity between the C-terminal regulatory domain of fungal ATP sulfurylase and fungal APS kinase, a homodimer that undergoes a temperature-dependent, reversible dissociation of subunits over a narrow temperature range. Wild-type ATP sulfurylase yielded hyperbolic velocity curves between 18 and 30 degrees C. Increasing the assay temperature above 30 degrees C at a constant pH of 8.0 increased the cooperativity of the velocity curves. Hill coefficients (n(H)) up to 1.8 were observed at 42 degrees C. The bireactant kinetics at 42 degrees C were the same as those observed at 30 degrees C in the presence of PAPS, the allosteric inhibitor. In contrast, yeast ATP sulfurylase yielded hyperbolic plots at 42 degrees C. The P. chrysogenum mutant enzyme, C509S, which is intrinsically cooperative (n(H) = 1.8) at 30 degrees C, became more cooperative as the temperature was increased yielding n(H) values up to 2.9 at 42 degrees C. As the temperature was decreased, the cooperativity of C509S decreased; n(H) was 1.0 at 18 degrees C. The cumulative results indicate that increasing the temperature increases the allosteric constant, L, i.e., promotes a shift in the base-level distribution of enzyme molecules from the high MgATP affinity R state toward the low MgATP affinity T state. As a result, the enzyme displays a true "temperature optimum" at subsaturating MgATP. The reversible temperature-dependent transitions of fungal ATP sulfurylase and APS kinase may play a role in energy conservation at high temperatures where the organism can survive but not grow optimally.     

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)     

Adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.

[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.     

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.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

The "regulatory" sulfhydryl group of Penicillium chrysogenum ATP sulfurylase. Cooperative ligand binding after SH modification; chemical and thermodynamic properties

ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Crystal structure of ATP sulfurylase from Penicillium chrysogenum: insights into the allosteric regulation of sulfate assimilation

ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.     

Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis

Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C.     

Adenosine di- and triphosphate transport in mitochondria. Role of the amidine region for substrate binding and transport

A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.     

Nucleoside triphosphate binding and hydrolysis by histone H1.

We present here further evidence supporting that histone H1 contains a nucleotide binding site interacting e.g. with ADP, ATP, GDP and GTP. The finding is in accordance with the previous observation that nucleotides modulate recognition of DNA by H1. Most interestingly, H1 appears to be capable of hydrolyzing NTPs and incorporating phosphate to exogenous proteins. The mode of nucleotide action on H1 may be considered highly analogous to that of GTPases. Nuclear receptors may thus act through mechanisms similar to those for receptors on the plasma membrane.     

Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro

Evidence indicates that a large portion of the facilitative glucose transporter isoform GLUT1 in certain animal cells is kept inactive and activated in response to acute metabolic stresses. A reversible interaction of a certain inhibitor molecule with GLUT1 protein has been implicated in this process. In an effort to identify this putative GLUT1 inhibitor molecule, we studied here the effects of adenosine and adenosine triphosphate (ATP) on the binding of D-glucose to GLUT1 by assessing their abilities to displace cytochalasin B (CB), using purified GLUT1 in vesicles. At pH 7.4, adenosine competitively inhibited CB binding to GLUT1 and also reduced the substrate binding affinity by more than an order of magnitude, both with an apparent dissociation constant (K(D)) of 3.0 mM. ATP had no effect on CB and D-glucose binding to GLUT1, but reduced adenosine binding affinity to GLUT1 by 2-fold with a K(D) of 30 mM. At pH 3.6, however, ATP inhibited the CB binding nearly competitively, and increased the substrate binding affinity by 4--5-fold, both with an apparent K(D) of 1.22 mM. These findings clearly demonstrate that adenosine and ATP interact with GLUT1 in vitro and modulate its substrate binding affinity. They also suggest that adenosine and ATP may regulate GLUT1 intrinsic activity in certain cells where adenosine reduces the substrate-binding affinity while ATP increases the substrate-binding affinity by interfering with the adenosine effect and/or by enhancing the substrate-binding affinity at an acidic compartment.     

Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...     

Kinetic studies of the choline acetyltransferase reaction using isotope exchange at equilibrium

Isotope exchange at equilibrium has been used to study the kinetic mechanism of the choline acetyltransferase reaction. The choline-acetylcholine, acetyl-CoA-acetylcholine, and CoA-acetyl-CoA exchange patterns are qualitatively consistent with a Theorell-Chance mechanism. However, quantitative differences are observed when the experimental results are compared to theoretical fits of the data for a Theorell-Chance mechanism. It is concluded that the kinetic mechanism of the choline acetyltransferase reaction can best be described as a random Theorell-Chance mechanism in which a low but finite amount of ternary complex exists.