Studies on a 3beta-hydroxysteroid sulphotransferase from rat liver.

A steroid sulphotransferase (EC 2.8.2.2) was partially purified from female rat liver. The enzyme was active towards the substrates, dehydroepiandrosterone, epiandrosterone and pregnenolone but was inactive towards oestrogens, cholesterol and ergocalciferol. A pH optimum of 5.0 was recorded but the enzyme was unstable at low pH. The enzyme was stimulated slightly by the addition of reducing agents and inhibited by p-chloromercuribenzoate and HgCl2. Crude enzyme activity was markedly stimulated by divalent cations but this effect was not observed with purified enzyme. A Km of 13 muM was calculated for the donor substrate 3'-phosphoadenylyl sulphate and the acceptor substrate, dehydroepiandrosterone had a Km value of 6 muM. The enzyme appeared to be highly susceptible to product inhibition by adenosine 3', 5'-diphosphate.     

INHIBITION OF RAT BRAIN PHENOL SULPHOTRANSFERASE IN VITRO BY NORADRENALINE and DOPAMINE METABOLITES1

Abstract— Kinetic parameters of the sulphotransferase reaction in rat brain were investigated in vitro at pH 7.4. Evidence is presented that the enzyme phenol sulphotransferase (EC 2.8.2.1) can be assayed with 4-methylumbelliferone or 3-methoxy-4-hydroxyphenylethyleneglycol as the substrate. Both assays give identical V_max values, whereas K_m values are 0.026 mm and 0.039 mm , respectively. Normetanephrine, metanephrine and the catecholamines adrenaline and dopamine, having a positive charge on the side chain at pH 7.4, do not inhibit 4-methylumbelliferone and 3-methoxy-4-hydroxyphenylethy-leneglycol sulphotransferase at this pH. Their deaminated metabolites 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenethanol and 3-methoxy-4-hydroxyphenylacetic acid inhibit both the enzyme activities. The type of inhibition is noncompetitive with the exception of 3-methoxy-4-hydroxy-phenylethyleneglycol, which is a competitive inhibitor of 4-methylumbelliferone sulphation. 3-Methoxy-4-hydroxy-mandelic acid does not inhibit the enzyme activities. It is concluded that the catecholamines themselves are not sulphated by rat brain in vitro at pH 7.4.     

Kinetic studies of the phenol sulphate-phenol sulphotransferase of Aspergillus oryzae.

Arylsulphatase II of Aspergillus oryzae exhibits both hydrolytic and sulphotransferase activities. The kinetic data suggest the formation of an intermediate covalent enzyme-sulphate complex with transfer of sulphate from donor to acceptor proceeding via a Ping Pong mechanism. The unusual kinetic behaviour when 2-hydroxy-5-nitrophenyl sulphate is the substrate is also consistent with this mechanism.     

Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.

The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.     

The sulphation of chondroitin sulphate in embryonic chicken cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.     

Hymenolepis diminuta: lack of sulphate activation and sulphotransferase activity

There is no evidence that Hymenolepis diminuta can carry out sulphoconjugation reactions. Neither whole worms nor worm extracts were able to sulphate 4-methylumbelliferone. No sulphotransferase activity could be demonstrated in H. diminuta using a variety of substrates, nor was H. diminuta capable of synthesising the sulphate donor 3'-phosphoadenosine-5'-phosphosulphate from ATP and inorganic sulphate. Possible alternative sources of active sulphate in this parasite are discussed.     

Thiol-dependent changes in the properties of rat liver sulphotransferases

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.     

The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives

1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37 degrees C the K(m) values for l-tyrosine methyl ester and adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate are 0.3mm and 8nm respectively. The K(m) value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the K(m) and V(max.) values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30 degrees C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate but not by l-tyrosine methyl ester.     

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.     

Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

Enzymatic sulphation of some galactose- containing sphingolipids in developing rat brain

Abstract— Cerebroside sulphotransferase has been found to catalyze the transfer of sulphate from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to both the α-hydroxy fatty acid galactosylceramides and the nonhydroxy fatty acid galactosylceramides. The sulphotransferase has a higher affinity for the α-hydroxy fatty acid galactosylceramides than for the nonhydroxy fatty acid galactosylceramides and will also use lactosylceramide as an acceptor for the transfer of sulphate from PAPS. A second sulphotransferase, PAPS: psychosine sulphotransferase, is also present in the developing rat brain and will catalyse the transfer of sulphate from PAPS to galactosylsphingosine and lactosylsphingosine. The sulphate moiety was determined to be on the galactose and most likely on the 3′ position giving a proposed structure of: 3-O-SO₄-galactosylsphingosine. The possible role of this later pathway in the synthesis of sulphogaiactosylceramide remains to be elucidated.     

Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues

The formation of sulpho-conjugates of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds was examined in preparations of rat tissues. Liver high-speed-supernatant preparations readily transferred sulphate from adenosine 3'-phosphate 5'-sulphato-phosphate to dopamine under standard conditions. The main product was identified as the 3-O-sulphate. The preparation also sulphated the 3- and 4-methoxy derivatives but to a lesser extent (44% and 95% respectively) relative to dopamine. Brain preparations possessed only half the activity of liver but formed both the 3- and 4-O-sulphates in the molar ratio of 1.7:1. l-3,4-Dihydroxyphenylalanine (l-dopa) in both tissue preparations did not yield any significant amount of sulpho-conjugate when the dopa decarboxylase present was inhibited. The sulphotransferase activity of preparations was doubled in the presence of dithiothreitol and it was concluded that l-tyrosine methyl ester sulphotransferase was the enzyme involved. A method for the preparation of authentic dopamine 3-O-sulphate and 4-O-sulphate was developed.     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

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.     

Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.31) was purified 1800-fold from rabbit liver by detergent extraction, differential solubility in polyethylene glycol and (NH4)2SO4, and column chromatography on DEAE-Sephacel, phenyl-Sepharose, CM(carboxymethyl)-Sepharose, Affi-Gel Blue, and Ultrogel AcA-34. The enzyme had a native Mr of 74,000 and appeared to be a homodimer with subunit Mr = 34,000. The enzyme was specific for NAD+. It oxidized both S-3-hydroxyisobutyrate and R-3-hydroxyisobutyrate, but the kcat/Km was approximately 350-fold higher for the S-isomer. Steady state kinetic analysis indicates an ordered Bi Bi reaction mechanism with NAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate between pH 7.0 and 11.5 with optimal activity between pH 9.0 and 11.0. The enzyme apparently does not have a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and NAD+ binding sites since inhibition by sulfhydryl-binding agents was differentially blocked by each substrate. The enzyme is highly sensitive to product inhibition by NADH which may play an important physiological role in regulating the complete oxidation of valine beyond the formation of 3-hydroxyisobutyrate.     

Studies in vitro on the involvement of O-sulphate esters in the formation of O-methylated 3,4-dihydroxybenzoic acid by rat liver.

The involvement of O-sulphate esters in the directed O-methylation was investigated in vitro with a dialysed "high-speed' supernatant from rat liver as the enzyme preparation and the catechol compound 3,4-dihydroxybenzoic acid as the substrate. The enzyme reactions involved were studied separately with the O-methylated and O-sulphated derivatives. The rate of hydrolysis by arylsulphatase was 14.5 nmol/min per mg of protein for 3-methoxy-4-sulphonyloxybenzoic acid and 10.1 nmol/min per mg of protein for 4-methoxy-3-sulphonyloxybenzoic acid. The sulphotransferase activity towards the guaiacols 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid was 570pmol of 4-O-sulphated and 350pmol of 3-O-sulphated product formed/min per mg of protein. The 3-O- and 4-O-sulphate esters of 3,4-dihydroxybenzoic acid could not serve as substrates for the catechol O-methyltransferase reaction. When either ester was incubated in the presence of S-adenosyl-L-methionine, but without the arylsulphatase inhibitor KH2PO4, 3,4-dihydroxybenzoic acid was formed, which was subsequently O-methylated in a meta/para ratio of 4.6. It is concluded that O-methylation can precede O-sulphation but that O-sulphation prevents further metabolism by O-methylation. Also O-sulphate esters do not have a directing effect on O-methylation. From the study of the simultaneous action of sulphotransferase and catechol O-methyltransferase on 3,4-dihydroxybenzoic acid we conclude that O-sulphation and O-methylation proceed independently of each other under the assay conditions used, both directed preferentially to the 3-hydroxy group.     

Aminoglycoside 2'-phosphotransferase type IIIa from Enterococcus

Aminoglycoside 2'-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2'-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2'-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2'-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.     

Donor activation in the T4 RNA ligase reaction

T4 RNA ligase catalyzes the adenylation of donor oligonucleotide substrates. These activated intermediates react with an acceptor oligonucleotide which results in phosphodiester bond formation and the concomitant release of AMP. Adenylation of the four common nucleoside 3',5'-bisphosphates as catalyzed by T4 RNA ligase in the absence of an acceptor oligonucleotide has been examined. The extents of product formation indicate that pCp is the best substrate in the reaction and pGp is the poorest. Kinetic parameters for the joining reaction between the preadenylated nucleoside 3',5'-bisphosphates, A(5')pp(5')Cp or A(5')pp(5')Gp, and a good acceptor substrate (ApApA) or a poor acceptor substrate (UpUpU) have been determined. The apparent Km values for both preadenylated donors in the joining reaction are similar, and the reaction velocity is much faster than observed in the overall joining reaction. The nonnucleotide adenylated substrate P1-(5'-adenosyl) P2-(o-nitrobenzyl) diphosphate also exhibits a similar apparent Km but reacts with a velocity 80-fold slower than the adenylated nucleoside 3',5'-bisphosphates. By use of preadenylated donors, oligonucleotide substrates can be elongated more efficiently than occurs with the nucleoside 3',5'-bisphosphates.