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.

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.

ATP sulfurylase from filamentous fungi: which sulfonucleotide is the true allosteric effector?

Fungal ATP sulfurylase has been reported to be allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the product of adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the sulfate activation sequence. However, the affinity of ATP sulfurylase for its immediate product, APS, is 1000 times higher than that for PAPS. Moreover, each sulfurylase subunit contains two sulfonucleotide binding sites (the catalytic site and a C-terminal, APS kinase-like allosteric site). Consequently, the possibility that the cooperative effects were caused solely by trace levels of APS, or by APS acting in concert with PAPS could not be dismissed. To identify the true allosteric effector, the molybdolysis reaction kinetics in the absence and in the presence of APS kinase were compared. The rationale was that in the absence of APS kinase, submicromolar levels of APS would be generated from contaminating SO(2-)4 present in the assay components, while in the presence of APS kinase, any APS formed would be converted to PAPS. The results were as follows: In the presence of added APS kinase, the initial velocity versus [MgATP] or versus [MoO(2-)4] plots at 100 microM PAPS were clearly sigmoidal as was the velocity versus [PAPS] plot at subsaturating substrate levels. Hill coefficients were in the range of 2 to 3. Also, low concentrations of S2O(2-)3offn inhibitor competitive with MoO(2-)4, activated the reaction at high PAPS and low substrate levels. These results are consistent with PAPS serving as a classical allosteric inhibitor. Although APS kinase should be superfluous to the molybdolysis reaction, the omission of this enzyme from assay mixtures resulted in rates that were higher, the same as, or lower than the corresponding "plus APS kinase" rates, (depending on the fixed level of substrates and PAPS). Additionally, the "minus APS kinase" velocity curves were less sigmoidal and, in some cases, nearly hyperbolic. The effect of APS kinase was shown to be catalytic in nature. If the data are analyzed in terms of the concerted transition (symmetry) model for allosteric enzymes, the cumulative experimental results indicate that PAPS is the true allosteric inhibitor of fungal ATP sulfurylase, binding preferentially to the T-state allosteric site (or to the allosteric site of the R state inducing the R --> T transition), while APS binds preferentially to the R state, probably as a competitive product inhibitor at the catalytic site. If it is assumed that occupancy of the allosteric site by any ligand that fits would induce the R --> T transition, then the results suggest that the allosteric site has evolved to have a higher affinity for PAPS than for APS (in contrast to real APS kinase). Computer-assisted simulations allowing for APS and PAPS binding to both the catalytic and regulatory sites of the hexameric enzyme yielded results that nearly duplicated the experimental curves.

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 5'-phosphosulfate (APS) kinase: diagnosing the mechanism of substrate inhibition.

Adenosine 5'-phosphosulfate (APS) kinase is subject to strong substrate inhibition by APS. The inhibition has been variously reported to be uncompetitive with respect to MgATP (resulting from the formation of a dead-end E. APS. MgADP complex) or competitive with MgATP (resulting from the formation of a dead-end E. APS complex). It is shown that these two types of substrate inhibition can be differentiated for ordered kinetic mechanisms by simple inspection of the v versus [APS] plots at different fixed concentrations of MgATP. Linear diagnostic plots are unnecessary. One diagnostic feature is the changing position of [APS]opt, the concentration of APS that yields the peak velocity. In the uncompetitive system, [APS]opt decreases asymptotically to a limit as the fixed [MgATP] is increased, while in the competitive system, [APS]opt increases continuously as the fixed [MgATP] is increased. A second (and more easily discerned) diagnostic feature is that, at any given inhibitory level of APS, enzyme activity relative to the velocity at [APS]opt (v/vopt) decreases as the fixed [MgATP] is increased in the uncompetitive system, while in the competitive system the relative activity increases as the fixed [MgATP] is increased. Normalized plots of v/vopt versus [APS] clearly display these distinguishing characteristics. The method confirmed that Penicillium chrysogenum APS kinase exhibits uncompetitive inhibition by APS.