A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Lowe, Gordoǹ; Sproat, Brian S.; Tansley, Gaynor; Cullis, Paul M.

doi:10.1021/bi00274a037

Cited by 17 publications

(5 citation statements)

References 40 publications

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“…The conditions employed for the desulfurization of (S'P)-[a-1802]dATPaS in [170] water were chosen carefully to minimize depurination due to low pH conditions generated during the course of the reaction. The use of low pH during the reaction, however, is necessary in order to avoid complications caused by the neighboring group participation by the terminal phosphate group during the hydrolysis of the bromothiophosphoryl intermediate at a higher pH Lowe et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

Stereochemical course of the 3' .fwdarw. 5'-exonuclease activity of DNA polymerase I

Gupta¹,

Benkovic²

1984

Biochemistry

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(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.

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Section: Discussionmentioning

confidence: 99%

Stereochemical course of the 3' .fwdarw. 5'-exonuclease activity of DNA polymerase I

Gupta¹,

Benkovic²

1984

Biochemistry

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“…(b) Elucidation of the absolute configuration of chiral DPPsE and chiral DPPsC, on the basis of a safe assumption that desulfurization proceeds with inversion of configuration (Connolly et al, 1982;Lowe et al, 1982Lowe et al, , 1983Potter et al, 1983;Sammons & Frey, 1982;Senter et al, 1983), makes it possible to interpret the results of biochemical study by use of chiral thiophospholipids in a stereochemical way. For example, it is now clear that phospholipase A2 is specific to the RP isomer of DPPsC and DPPsE whereas phospholipases C and D are specific to the Sp isomer.…”

Section: Discussionmentioning

confidence: 99%

“…The configuration of chiral DPPsC and chiral DPPsE can therefore be elucidated by conversion to [lsO]DPPE by procedures of known stereochemistry. It has been shown that desulfurization of various nucleoside phosphorothioates in H2180 mediated by bromine, cyanogen bromide, or Nbromosuccinimide proceeds with inversion of configuration at phosphorus (Connolly et al, 1982;Lowe et al, 1982Lowe et al, , 1983Potter et al, 1983;Sammons & Frey, 1982;Senter et al, 1983). Such a reaction can therefore be used to correlate the configuration of chiral thiophospholipids with that of [lsO]- with no detectable products.…”

Section: Conversion Of Dppscmentioning

confidence: 99%

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Jiang¹,

Shyy²,

Tsai³

1984

Biochemistry

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Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.

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“…Adenosine (R)-5'-[a-170]triphosphate was prepared as described previously [26,27], and was shown to contain 6 atom % l60, 42 atom % 170, and 52 atom % l80 by 31P-NMR spectroscopy and from the known I7O: l80 ratio (1 : 1.223) in the [170]water used in the synthesis; the sample contained 6 % of the (S)-isomer 126, 271. …”

Section: Methodsmentioning

confidence: 99%

The Stereochemical Course of Nucleotidyl Transfer Catalysed by NAD Pyrophosphorylase

Lowe

Tansley

1983

European Journal of Biochemistry

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NAD pyrophosphorylase catalyses nucleotidyl transfer from adenosine (R)-~'- [E-' 70]triphosphate to nicotinamide mononucleotide with inversion of configuration at the a-P giving (S)- ['70]NAD'. The simplest interpretation of this observation is that the adenylyl group is transferred directly from ATP to the co-substrate by an 'in line' mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD ' regio-specifically at the adenylyl terminus of the pyrophosphate bond.NAD pyrophosphorylase catalyses nucleotidyl transfer from MgATP to nicotinamide mononucleotide (NMN) to form NAD' [I].This reaction is the final step in the main biosynthetic pathway to NAD' in eukaryotic cells [2] and it is particularly noteworthy that NAD pyrophosphorylase is the only enzyme in the pathway that is located in the cell nucleus [3 -51. It has been suggested that the nuclear location of NAD pyrophosphorylase is related to the involvement of NAD+ in the regulation of cellular activity [6] and a correlation between NAD pyrophosphorylase activity and mitosis has been observed [7, 81; there are also numerous examples of a correlation between NAD pyrophosphorylase activity and DNA synthesis [4,. This suggestion was supported by the discovery of a chromosomal protein, poly(ADP-ribose) synthetase [13 -151, which uses NAD' as a substrate for the post-translational modification of nuclear proteins [16,17], including histones [I 8, 191. Although the precise physiological role of this enzyme remains unclear, there is evidence that poly(ADP-ribosy1)ation is involved in DNA replication, mitosis, transcription and the repair of damaged DNA [17, 20-231.NAD pyrophosphorylase activity is very low in tumour cells when compared with normal adult cells and is probably close to the critical level for cell survival [4, 81. Consequently NAD pyrophosphorylase has been regarded as a potential target site for cancer chemotherapy [S], since the minimum inhibitory concentration lethal to tumour cells is not likely to cause serious damage to normal cells. Since a knowledge of the mechanism of action of this enzyme and the juxtaposition of the substrates when bound to the enzyme would undoubtedly assist in the design of a specific inhibitor, we have investigated the stereochemical course of nucleotidyl transfer catalysed by .NAD pyrophosphorylase.N AD pyrophosphorylase catalyses exchange between ATP and [32P]PPi in the presence of NMN; in the absence of NMN Enzyme. NAD pyrophosphorylase or NMN adenylyltransferase (EC 2.7.7.1).only a small amount of exchange occurs and the reasonable conclusion was made that there is no support for an adenylylenzyme intermediate [24]. However the possibility of an induced conformational change of the enzyme on binding NMN leading to a more facile adenylyl transfer to the putative adenylyl-enzyme intermediate is difficult to rule out [25]. We have undertaken therefore to determine the stereochemical course of the nucleotidyl transfer catalysed by NAD pyrophosphorylase using adenosine (R)-5'-[a-170]triphosphate [26,...

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A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Cited by 17 publications

References 40 publications

Stereochemical course of the 3' .fwdarw. 5'-exonuclease activity of DNA polymerase I

Stereochemical course of the 3' .fwdarw. 5'-exonuclease activity of DNA polymerase I

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

The Stereochemical Course of Nucleotidyl Transfer Catalysed by NAD Pyrophosphorylase

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