Purification of spermine synthase from bovine brain by spermine—Sepharose affinity chromatography

Rl, Pajula; Raina, Aarne; Kekoni, J

doi:10.1016/0014-5793(78)80319-6

Cited by 22 publications

(5 citation statements)

References 10 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…0,l ml; 0.1 M potassium phosphate buffer (PH 7,4)$ 1 m&I spermidine; 0.04 mM decarboxylated S&anosyl-methionlne; 5 mM dithiothreitol; 0.05-0.1 @g enzyme protein. Protein was measured as in [3].…”

Section: Introductionmentioning

confidence: 99%

Methylthioadenosine, a potent inhibitor of spermine synthase from bovine brain

1979

Self Cite

View full text Add to dashboard Cite

Section: Introductionmentioning

confidence: 99%

Methylthioadenosine, a potent inhibitor of spermine synthase from bovine brain

1979

Self Cite

View full text Add to dashboard Cite

“…Spermidine synthase is reported to be sensitive to product inhibition by MTA with an inhibition constant of 50 µM for rat spermidine synthase (9). Mammalian spermine synthase is more sensitive to MTA, with a K i value of 0.3 µM (10). Inhibition of MTAN is therefore expected to inhibit polyamine biosynthesis and the salvage pathways for adenine and methionine.…”

mentioning

confidence: 99%

Transition State Structure of 5‘-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli and Its Similarity to Transition State Analogues

et al. 2005

View full text Add to dashboard Cite

Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5'-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1'-(3)H, 1'-(14)C, 2'-(3)H, 4'-(3)H, 5'-(3)H, 9-(15)N, and Me-(3)H(3) were 1.160 +/- 0.004, 1.004 +/- 0.003, 1.044 +/- 0.004, 1.015 +/- 0.002, 1.010 +/- 0.002, 1.018 +/- 0.006, and 1.051 +/- 0.002, respectively. The large 1'-(3)H and small 1'-(14)C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (D(N)A(N)) (S(N)1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 A from the anomeric carbon. The small beta-secondary 2'-(3)H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'-H2' dihedral angle of 53 degrees at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-(3)H KIE is due to hyperconjugation between the lone pair (n(p)) of O3' and the antibonding (sigma) orbital of the C4'-H4' group, and the methyl-(3)H(3) KIE is due to hyperconjugation between the n(p) of sulfur and the sigma of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.

show abstract

“…Spermidine and spermine synthases (aminopropyltransferases) were purified from rat ventral prostate as in [20,21]. Extracts containing both enzyme activities were fractionated into spermidine synthase and spermine synthase preparations by the treatment with ammonium sulfate followed by DEAE-cellulose and Ultrogel ACA 34 column chromatography.…”

Section: Preparation Of Spermidine and Spennine Synthasesmentioning

confidence: 99%