Irwin H. Segel

Specificity of transport processes for sulfur, selenium, and molybdenum anions by filamentous fungi☆

1. 1. Inorganic SO42−, S2O32−, SeO42− and MoO42− enter mycelia of Penicillium and Aspergillus species by a common energy-, temperature-, pH-, and concentration-dependent permease. Evidence for a single permease is as follows: (a) All four anions exhibit reciprocal competitive inhibition. (b) Mycelia grown on sulfur sources that repress the sulfate permease (e.g.), l-methionine) show similar low S2O32−, SeO42−, and MoO42− transport rates. Mycelia grown on sulfur sources that derepress the sulfate permease (e.g.), l-djenkolic and l-cysteic acids) transport all four anions rapidly. (c) Sulfur starvation results in coincident derepression of SO42−, S2O32−, SeO42, and MoO42 transport to at least 40 times the base level. Throughout the derepression period the SeO42 /SO42, MoO42− and SeO42−/MoO42 transport rate ratios remain constant. (d) A mutant defective in SO42− transport was equally defective in SeO42 and MoO42− transport. 2. 2. In addition to the sulfate (thiosulfate, selenate, molybdate) permease the mycelia possess distinct permeases for SO32− and S4O62−. The sulfite and tetrathionate permeases are under metabolic control by some intracellular sulfur-containing metabolite. 3. 3. Inorganic S2O32− enters the mycelium of a sulfate (thiosulfate) permease-negative mutant at 1–5% of the wild-type rate at low extracellular S2O22 concentrations. S2O32− transport by the mutant is not inhibited by SO42−. The vmax for S2O32− transport by the mutant is almost the same as that of the sulfate permease-positive parent (about 2–3 μmoles/g per min). The Km value, however, is about 30-fold higher (2 mM vs. 59 μM). The tetrathionate permease may be responsible for S2O32-transport in the mutant under standard assay conditions. However, S2O32 is unstable at the pH values of most fungal cultures. Consequently, most of the sulfur incorporated from S2O3--containing media in long-term growth studies is probably in the form of breakdown products (SO32-, SO42-, S2-). 4. 4. S2- uptake is 2,4-dinitriphenol- and azide-sensitive, but shows a low Q10 (1.15 vs. 2.1 for the sulfate permease), is non-saturable, and does not depend on the degree of sulfur sufficiency of the mycelium. 5. 5. No SCN- uptake could be detected.

Specificity and regulation of methionine transport in filamentous fungi

Abstract l -Methionine uptake by mycelium of Penicillium chrysogenum (under physiological conditions) is mediated by two distinctly different, independently regulated, stereospecific membrane transport systems (permeases). One of the systems is relatively specific for l -methionine. The other is a general (nonspecific) amino acid permease. The specific l -methionine permease develops upon sulfur-starvation. l -methionine transport by sulfur-starved mycelium is at least ten times more rapid than by sulfur-sufficient mycelium at low external methionine concentrations and is strongly inhibited only by analogs and derivatives of l -methionine (e.g., l -ethionine, ethionine and methionine sulfoxides, sulfones, sulfoximines, methyl and ethyl esters, and homocysteine). Nonsulfur amino acids show little or no inhibition. Sulfur-starvation has little effect on the rates of transport of nonsulfur amino acids. The specific system is (a) pH-dependent (optimum at pH 6 with 50% of maximum velocity at pH 3 and 9), (b) temperature-dependent ( Q 10 of 2 between 5 ° and 35 °), (c) energy-dependent (inhibited by 2,4-dinitrophenol, azide, and p -chloromercuribenzoate), (d) relatively insensitive to variations in the ionic strength of the incubation medium, (e) independent of metal ions for activity, and (f) independent of concurrent protein synthesis. Transport is concentration-dependent showing saturation kinetics. The V max in sulfur-sufficient and sulfur-deficient mycelia is 1–3 μmoles/gm-minute. Sulfur starvation results in a decrease in the K m for l -methionine from 10 −3 to 10 −5 m . Refeeding sulfur-starved mycelia with various sulfur compounds results in a rapid increase in the K m back to the sulfur-sufficient level. These results suggest that the specific l -methionine permease is regulated primarily by feedback inhibition-deinhibition rather than by repression-derepression. Inhibition studies suggest that the minimum structural requirements for strong interaction of a compound with the specific methionine permease include (a) a sulfur atom containing no ionizable groups, (b) two methylene groups between the sulfur atom and the α-carbon, (c) an unsubstituted l -α-amino group, (d) an unsubstituted α-hydrogen, and (e) a primary carbonyl group. The nonspecific transport system is deinhibited, or derepressed (or both), upon nitrogen starvation. l -methionine transport by nitrogen-deficient mycelia is 20–100 times more rapid than by nitrogen-sufficient mycelia at low external methionine concentrations, and is strongly inhibited by almost all neutral and basic l -α-amino acids. Nitrogen starvation results in an increase in the rates of transport of all other amino acids tested ( l -tryptophan, l -phenylalanine, l -leucine, l -serine, l -glutamic acid, and l -lysine).

The inorganic sulfate transport system of Penicillium chrysogenum

Abstract Inorganic sulfate enters the mycelium of Penicillium chrysogenum against an apparent concentration gradient by a temperature-, energy-, pH-, and concentration-dependent transport system that is regulated by the degree of sulfur-deficiency of the mycelium. Sulfate transport can take place in the absence of sulfate reduction and organic sulfur formation. The rapid conversion of sulfate to organic sulfur in the absence of N -ethylmaleimide suggests that the newly-transported 35 SO 4 −− does not completely equilibrate with the pre-existing sulfate pool. Only about half of the transported sulfate could be removed by exhaustive washing or exchanged with unlabeled extracellular sulfate. The kinetics of transport and exchange also suggest the presence of two distinct intracellular sulfate pools. Inorganic thiosulfate, a suggested intermediate and overflow product of fungal sulfate reduction is an extracellular inhibitor of sulfate transport. Sulfite is an extracellular inhibitor at pH 5 but not at pH 6.5. Methionine and cysteine are not extracellular inhibitors of sulfate transport, but are rapidly converted intracellularly to a feedback inhibitor. The V max and apparent K m of the sulfate transport system (in the presence of NEM) are 0.5 – 1.0 μ mole/gmminute and 3–7 × 10 −5 m , respectively, at pH 6.5. Inorganic selenate inhibits sulfate transport and is taken up by the mycelium via the sulfate transport system. In the absence of net transport, sulfate will still adsorb to the mycelial surface very rapidly (or penetrate into a free space) by a DNP- and temperature-insensitive process.

Purification and properties of glycogen phosphorylase from Escherichia coli

Abstract Glycogen phosphorylase and maltodextrin phosphorylase were purified 1100-fold and 200-fold respectively from cell-free extracts of Escherichia coli, K-12. The optimum pH of the glycogen phosphorylase is 6.7–6.9 and the equilibrium favors glycogen synthesis from glucose-1-phosphate. The spectrum of the enzyme suggests that it contains pyridoxal phosphate. Heavy metals and pCMB are inhibitory. The Km values for glucose-1-phosphate and glycogen are 10−3 m and 0.67% w v , respectively in the absence of AMP and 2.9 × 10−3 m and 0.46% w v , respectively in the presence of 5 × 10−3 m AMP. AMP increased the Vmax of the reaction slightly when glycogen was not saturating. Glucose (Ki = 2.5 × 10−>m), ADPG (Ki = 0.7 × 10−>m), TDPG (Ki = 10−m), and UDPG (Ki = 2 × 10−m) were competitive inhibitors with respect to glucose-1-phosphate. Glucose-1-phosphate (at > 2 × 10− m ) was also inhibitory.

Escherichia coli polyglucose phosphorylases.

Abstract Escherichia coli , K-12, possesses two distinct polyglucose (oligoglucoside) phosphorylases (α-1,4-glucan: orthophosphate glucosyltransferase, E.C. 2.4.1.1). The two enzymes may be separated by ammonium sulfate fractionation or by DEAE-cellulose column chromatography. One of the enzymes is a maltodextrin phosphorylase. This enzyme is present in glucose-grown cells but is induced to a 10-fold higher level in maltose-grown cells. The maltodextrin phosphorylase shows a decided preference for short chain dextrins over glycogen as the polyglucose acceptor. The second enzyme is a constitutive glycogen phosphorylase. The level of the glycogen phosphorylase is independent of the carbon source used for growth. The glycogen phosphorylase shows higher activity with glycogen than with dextrin. The maltodextrin phosphorylase is much more heat stable than the glycogen phosphorylase. Both enzymes are stimulated slightly by AMP and strongly inhibited by heavy metals and pCMB. The glycogen phosphorylase shows a 4-fold activation by NaF and a 10-fold activation by Na 2 SO 4 . Sulfate activation is sigmoidal. The maltodextrin phosphorylase shows little or no salt activation.

Adenosine triphosphate sulfurylase from Penicillium chrysogenum equilibrium binding, substrate hydrolysis, and isotope exchange studies.

Abstract ATP sulfurylase from Penicillium chrysogenum was purified to homogeneity. The enzyme binds 8 mol of free ATP (Ks = 0.53 mM) or AMP (Ks = 0.50 mM) per 440,000 g. The results are consistent with our earlier report that the enzyme is composed of eight identical subunits of Mr 55,000 ( J. W. Tweedie and I. H. Segel, 1971, Prep. Biochem. 1, 91–117; J. Biol. Chem. 246, 2438–2446 ). In the absence of cosubstrates, the purified enzyme catalyzes the hydrolysis of MgATP (to AMP and MgPPi) and adenosine 5′-phosphosulfate (APS) (to AMP and SO42−). MgATP hydrolysis is inhibited by nonreactive sulfate analogs such as nitrate, chlorate, and formate (uncompetitive with MgATP). In spite of the hydrolytic reactions it is possible to observe the binding of MgATP and APS to the enzyme in a qualitative (nonequilibrium) manner. Neither inorganic sulfate (the cosubstrate of the forward reaction) nor formate or inorganic phosphate (inhibitors competitive with sulfate) will bind to the free enzyme in detectable amounts in the absence or in the presence of Mg2+, Ca2+, free ATP, or a nonreactive analog of MgATP such as Mg-α,β-methylene-ATP. Similarly, inorganic pyrophosphate (the cosubstrate of the reverse reaction) will not bind in the absence or in the presence of Mg2+ or Ca2+. The induced binding of 32Pi (presumably to the sulfate site) can be observed in the presence of MgATP. The results are consistent with the obligately ordered binding sequence deduced from the steady-state kinetics ( J. Farley et al., 1976, J. Biol. Chem. 251, 4389–4397 ) and suggest that the subsites for SO2−4 or MgPPi appear only after nucleotide cleavage to form E~AMP · MgPPi or E~AMP · SO4 complexes. The suggestion is supported by the relative values of Kia (ca. 1 m m for MgATP) and Kiq (ca. 1 α m for APS) and by the inconsistent value of k−1 calculated from V f K i a K m A (The value is considerably less than Vr) Purified ATP sulfurylase will also catalyze a Mg32PPi-MgATP exchange in the absence of SO42−. A 35SO42−-APS exchange could not be demonstrated in the absence or presence of MgPPi. This result was not unexpected: The rate of APS hydrolysis (or conversion to MgATP) is extremely rapid compared to the expected exchange rate. Also, the pool of APS at equilibrium is extremely small compared to the sulfate pool. The V values for molybdolysis, APS hydrolysis (in the absence of PPi), ATP synthesis (from APS + MgPPi), and Mg32PPi-MgATP exchange at saturating sulfate are all about equal (12–19 μmol × min−1 × mg of enzyme−1). The rates of Mg32PPi-MgATP exchange in the absence of sulfate, APS synthesis (from MgATP + sulfate), and MgATP hydrolysis (in the absence of sulfate) are considerably slower (0.10 – 0.35 μmol × min−1 × mg of enzyme−1). These results and the fact that k4 calculated from V r K i q K m Q is considerably larger than Vf suggest that the rate-limiting step in the overall forward reaction is the isomerization reaction E~AMP-SO2−4 → E APS . In the reverse direction the rate-limiting step may be SO2−4 release or isomerization of the E~AMP · MgPPi · SO42− complex. (The reaction appears to be rapid equilibrium ordered.) Reactions involving the synthesis or cleavage of APS are specific for Mg2+. Reactions involving the synthesis or cleavage of ATP will proceed with Mg2+, with Mn2+, and, at a lower rate, with Co2+. The results suggest that the enzyme possesses a Mg2+-preferring divalent cation (activator) binding site that is involved in APS synthesis and cleavage and is distinct from the MeATP or MePPi site. The equilibrium binding of about one atom of 45Ca2+ per subunit (possibly to the activator site) could be demonstrated (Ks = 1.4 mM).

Acidic and basic amino acid transport systems of Penicillium chrysogenum.

Abstract Penicillium chrysogenum possesses a relatively specific constitutive transport system for the basic amino acids l -lysine and l -arginine (Km = 6 × 10−6 m , Vmax = 1 μmole/g-min). This system has a low affinity for 2,4-diaminobutryate, l -ornithine, and l -histidine. The basic amino acid transport system can be assayed readily in nutrient-sufficient mycelia where a nonspecific amino acid transport system (Benko, P. V., Wood, T. C., and Segel, I. H., Arch. Biochem. Biophys.129, 498, 1969), which also transports basic amino acids, is extremely low in activity. Nutrient-sufficient mycelia also contain a low-level lysine-insensitive arginine transport system and a low-level arginine-insensitive lysine transport system. The basic amino acid transport system can be assayed in nitrogen-starved and in carbon-starved mycelia if a large excess of, e.g., l -leucine is added to the assay mixture in order to suppress transport of the labeled substrate by the nonspecific amino acid transport system. The acidic amino acids, l -glutamate and l -aspartate, are also substrates of the general amino acid transport system. However, the preferred ionic form is the uncharged species. l -Glutamate and l -aspartate are also transported by a distinct stereospecific acidic amino acid transport system that develops along with the nonspecific system during nitrogen starvation or carbon starvation. The acidic amino acid system can be assayed at pH 6.0 in the presence of a large excess of, e.g., l -leucine. These conditions minimize the transport of glutamate and aspartate by the nonspecific system. A distinct proline transport system has also been detected in nitrogen-starved and in carbon-starved mycelia. No distinct or quantitatively significant specific systems for aromatic amino acids or histidine were detected. The general and acidic amino acid transport systems are subject to feedback inhibition (“transinhibition”) by their intracellular substrates. The transinhibition is most evident under conditions in which the amino acid is transported much faster than it is metabolized. In this respect, l -α-aminoadipate is a better transinhibitor of the acidic system than the preferred substrates, aspartate and glutamate. The feedback inhibitor of the nonspecific system has been shown to be the accumulated substrate rather than NH4+.

Multiplicity and regulation of amino acid transport in Penicillium chrysogenum

Abstract Penicillium chrysogenum possesses a highly specific sulfur-regulated permease for l -methionine (Benko, Wood, and Segel, Arch. Biochem. Biophys.122, 783–804 (1967)). In addition, the mycelium has several other relatively specific amino acid permeases. For example, l -phenylalanine-14C transport by sulfur- and nitrogen-sufficient mycelium is not significantly inhibited by a 10-fold excess of unlabeled l -methionine, l -leucine, l -serine, or l -α-aminobutyric acid. Similarly, l -leucine-14C transport is not significantly inhibited by a 10-fold excess of any of the other four amino acids. Nitrogen-starvation results in the development of a nonspecific amino acid permease (Vmax for l -methionine, l -leucine, and l -phenylalanine ca. 10 μmoles/g-min). The development is prevented by actidione. Structural requirements for interaction with the nonspecific amino acid permease are: (a) an unsubstituted l -α-amino group, (b) a single α-hydrogen atom, (c) no secondary negatively charged group, and (d) an α-carbonyl group. The Km values of the nonspecific permease for l -phenylalanine and l -methionine are ca. 10−5 m . The Ki for l -leucine (with l -methionine as substrate) is 2.4 × 10−5 m ; the Ki for l -methionine (with l -leucine as substrate) is 1.4 × 10−5 m . Other characteristics of the permease are pH-dependence (optimum at 6), temperature-dependence (Q10 of 2–5 between 15 and 32 °), and energy-dependence (DNP and azide sensitive). The general amino acid permease is independent of monovalent and divalent metal ions and of concurrent protein synthesis for activity. Ammonia (NH4+) was the only non-amino acid inhibitor of the permease. The permease activity can be markedly and rapidly reduced by preloading the nitrogen-deficient mycelium with NH4+ or any one of several amino acids. The results suggest that the nonspecific amino acid permease is subject to feedback regulation by intracellular NH4+ and amino acid substrates.

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

Abstract When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l -leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l -leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase). The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.

Adenosine 5′-Phosphosulfate Kinase from Penicillium chrysogenum SITE-DIRECTED MUTAGENESIS AT PUTATIVE PHOSPHORYL-ACCEPTING AND ATP P-LOOP RESIDUES

The properties of Penicillium chrysogenum adenosine 5′-phosphosulfate (APS) kinase mutated at Ser-107 were examined. Ser-107 is analogous to a serine of the E. coli enzyme that has been shown to serve as an intermediate acceptor in the transfer of a phosphoryl group from ATP to APS. Replacement of Ser-107 with alanine yielded an active enzyme with kinetic characteristics similar to those of wild-type APS kinase. Another mutant form of the enzyme in which Ser-107 was replaced by cysteine was also active. Covalent modification of Cys-107 eliminated catalytic activity, and substrates protected against modification. Mutation of Ser-97, of Ser-99, of Thr-103, of Ser-104 to alanine, or of Tyr-109 to phenylalanine also yielded an active enzyme. The cumulative results indicate that Ser-107 may reside in the substrate binding pocket of fungal APS kinase, but neither it nor any nearby hydroxy amino acid serves as an obligatory phophoryl acceptor in the 3′-phosphoadenylylsulfate synthesis reaction. The results also indicate that the absence of a serine at position 478 in the APS kinase-like C-terminal region of fungal ATP sulfurylase does not account for the lack of APS kinase activity in that enzyme. However, mutating the ATP P-loop residues in APS kinase to those found in the analogous C-terminal region of fungal ATP sulfurylase eliminated enzyme activity.