On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.     

EPR Spectrum at 4, 9 and 35 GHz of hydrogenase from Chromatium vinosum. Direct evidence for spin-spin interaction between Ni(III) and the ironsulphur cluster

EPR spectra at 4, 9 and 35 GHz of hydrogenase isolated from Chromatium vinosum have been compared. The spectra at 4 and 35 GHz confirmed our earlier conclusions, made from observations at 9 GHz (Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309–316), that the irreversibly inactivated enzyme molecules in the preparation give rise to two EPR signals due to the independent non-interacting $\text{S =}\text{1}\text{2}$ systems of Ni(III) and a |3Fe-xS| cluster. It was observed that intact enzyme molecules show a complex EPR spectrum caused by a spin-coupled pair of Ni(III) and a |4Fe-4S|³⁺ cluster. The interaction energy is so weak (approx. 0.01 cm^?1) that the 35 GHz spectra of both the Ni(III) and the |4Fe-4S|³⁺ cluster have the appearance of rather normal $\text{S =}\text{1}\text{2}$ spectra with additional splittings as a result of the spin-spin interaction. At lower microwave frequencies, the spectra become increasingly complex but phenomenologically they behave as expected for an exchange-coupled pair of dissimilar ions. The distance between the two spin systems is estimated to be at the most 1.2 nm. The spin-relaxation rate of the Ni(III) ion is dramatically enhanced as a result of the coupling to the rapidly relaxing Fe-S cluster. The g values and so presumably also the ligand fields of Ni in intact and irreversibly inactivated enzyme molecules are identical. This suggests that the specific coordination of the nickel in the enzyme is not the only requirement for activity with artificial electron donors or acceptors, and that the presence of a nearby, intact |4Fe-4S|^3+(3+,2+) cluster might be another essential factor. From the g values and the probable function of Ni in the enzyme we propose, as a working hypothesis, that the nickel ion has five ligands provided by the protein in a square-pyramidal coordination.     

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from $\text{Desulfovibrio}\text{gigas}$is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ～2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.     

Immunological titration of rat liver glucose-6-phosphate dehydrogenase from animals fed high and low carbohydrate diets.

Recent work (Hizi and Yagil [1974] Eur. J. Biochem. $\text{45}$: 211–221, and Kelly $\text{et. al.}$ [1975] Fed. Proc. $\text{34}$: 881) suggests that the marked increase in rat liver glucose-6-phosphate dehydrogenase activity which is observed upon feeding an animal a high carbohydrate diet does not involve an increase in the total amount of enzyme present. In contrast, the data presented herein involving immunological titrations of rat liver glucose-6-phosphate dehydrogenase indicates that the increase in enzyme activity resulting from feeding a high carbohydrate diet does involve an increase in the total amount of enzyme present.     

Identification of inner- and outer-sphere reaction pathways in the reduction of iron-sulphur proteins with a chromium(II)-macrocycle complex

When parsley [2Fe-2S] and $\text{C. pasteurianum}$ 2[4Fe-4S] proteins in the normal oxidised state are reduced 1:1 with Cr(II) (15-aneN₄) (H₂O)₂²⁺ the Cr(III) product remains attached to the protein and reduction is by an inner-sphere mechanism. With $\text{Chromatium}$ high potential [4Fe-4S] protein and $\text{C. pasteurianum}$ rubredoxin the Cr(III) product is not attached to the protein and the mechanism is outer-sphere. Results are discussed in the context of protein crystallographic information. The Cr(III) product is not attached to the Fe₂S₂ core (extrusion experiments) or to the cysteinyl S-atoms (ESR). Negative patches close to the active site remain possible alternatives.     

Characterization of native 40S particles from krebs II mouse ascites tumor cells. I. Phosphorylation in vitro of non ribosomal proteins by a cAMP independent protein kinase.

In the presence of [γ³²-P] ATP or [γ³²-P] GTP 4 non ribosomal proteins (M_r 110,000; 105,000; 89,000 and 25,000) of the native 40S subunit became phosphorylated. The protein kinase responsible for this phosphorylation could be removed by treatment with 0.5$\text{M}$ KCl. Sucrose density gradient analysis showed that the endogenous enzyme activity sedimented with approx. 7.5S.     

Direct electrochemical oxidation of Clostridium pasteurianum ferredoxin: Identification of facile electron-transfer processes relevant to cluster degradation

Rapid oxidation processes relevant to the degradation of [4Fe4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential-pulse voltammograms of native [4Fe4S] ferredoxin showed two well-defined oxidation peaks corresponding to apparent E-values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe4Se] derivative for which peak positions were shifted. Square-wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super-oxidised’ [4Fe4S]³⁺ oxidation level, undergoes rapid degradation (t_{$\text{1}\text{2}$} < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (?100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FES(Se) substructure.     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

Alpha-chymotrypsin: effects of bicyclic substrate geometry and hydrophobicity on reactivity

The α-chymotrypsin-catalyzed hydrolysis rates of p-nitrophenyl cyclopentane-carboxylate (I), p-nitrophenyl indan-2-carboxylate (II), and p-nitrophenyl spiro-[4.4]nonane-2-carboxylate (III) were measured at pH 8.1 in 20% methanol. After correction for variations in reactivity owing to stereoelectronic effects inherent to the substrates, the deacylation rate constants (k_c)_n of I and II are not significantly different. In $\text{(}\text{k}{}_{\mathrm{c}}\text{K}{}_{\mathrm{m}}\text{)}{}_{\mathrm{n}}$ II is 50 times more reactive than I, which demonstrates that the aromatic ring of the former substrate contributes significantly to its reactivity. The nearly equal reactivities of II and III indicate that the enzyme is rather insensitive to the geometry of the nonester-bearing ring of these compounds.     

Crystallographic study of the iron-sulfur flavoprotein trimethylamine dehydrogenase from the bacterium W3A1

Large single crystals of trimethylamine dehydrogenase, containing both [4Fe-4S]²⁺ centers and covalently bound FMN, have been prepared by the macro seeding technique. The crystals are monoclinic, space group P2₁ with cell parameters $\text{a = 147.63}\text{A}\text{?}\text{, b = 71.96}\text{A}\text{?}\text{, c = 83.66}\text{A}\text{?}\text{and}\text{β = 97.64 °}$, and diffract to at least 2.0 Å resolution. There is one dimer of approximately 166,000 M_m per asymmetric unit. A 5.0 Å resolution anomalous scattering difference Patterson has been computed which shows the presence and position of two [4Fe-4S]²⁺ centers in the asymmetric unit. A self-rotation function computed at 6.0 Å resolution indicates a non-crystallographic 2-fold axis relating the two subunits. These results show trimethylamine dehydrogenase to be composed of two identical or very similar subunits each containing one [4Fe-4S]²⁺ center.     

Solubilization and anionic regulation of cerebral sedative/convulsant receptors labeled with [35S] tert-butylbicyclophosphorothionate (TBPS)

Binding activity for the cage convulsant [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABA_A/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [³⁵S]-$\text{t}$-butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Turnover of dihydrofolate reductase in rapidly dividing cells

The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [¹⁴C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2></mtext>}}$) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had $\text{t}{}_{\mathrm{<mtext>x1</mtext><mtext>2</mtext>}}$ values of approximately 3 and 9 hr.     

Explanation for the apparent inactivation of tyrosine aminotransferase in hepatoma cells by concanavalin A

Brief treatment of hepatoma cells in monolayer culture with concanavalin A causes a decrease in tyrosine aminotransferase specific activity that is thought to be a rapid, reversible inactivation of the enzyme (T.V. Gopalakrishnam and E.B. Thompson 1977 J. Biol. Chem. $\text{252}$, 2717–2725). We confirm this decrease, but attribute it to an increased leakage of cellular protein from concanavalin A-treated monolayer cultures during the harvesting procedure. If the cells are washed free of medium and lysed $\text{in}$,$\text{situ}$ by freezing and thawing them, or by treating them with buffer containing a nonionic detergent, equal amounts of tyrosine aminotransferase are found in concanavalin A-treated and untreated cells. If cells are harvested by scraping them from the substrate, some tyrosine aminotransferase is lost into the buffer used to collect the cells. Treatment of cells with concanavalin A markedly increases the amount of enzyme lost during this procedure, and results in a low enzyme content in the washed cells. No inactivation occurs, however, because the total amount of tyrosine aminotransferase present in the cell pellet and the wash buffer is equal for treated and untreated cells.     

Reversed-phase,ion-pair liquid chromatography of quaternary ammonium compounds : Determination of pyridostigmine,neostigmine and edrophonium in biological fluids

A reversed-phase, ion-pair liquid chromatographic method for the quantitative determination of quaternary acetylcholinesterase inhibitors is described. The method uses an ion-pair extraction to isolate the drugs from biological material prior to liquid chromatographic separation and online UV detection at 214 nm. Quantitation down to 5 ng/ml and within-day precision with coefficient of variation (C.V.) of 1.5% (n=10, $\text{x}$ = 100 ng/ml) for neostigmine, C.V., 1.7% (n=10, $\text{x}$ = 80 ng/ml) for pyridostigmine and C.V., 1.5% (n=10, $\text{x}$ = 100 ng/ml) for edrophonium have been achieved. The assay was designed for pharmacokinetic studies of these drugs in anesthetized patients.