Nuclear Overhauser effect studies of the conformations of tetraamminecobalt(III)-ATP free and bound to bovine heart protein kinase

Pr, Rosevear; Hn, Bramson; O’Brian, Catherine A.; Et, Kaiser; As, Mildvan

doi:10.1021/bi00283a021

Cited by 46 publications

(64 citation statements)

References 39 publications

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“…The initial slopes of the build-up curves gave direct measurements of the cross-relaxation rates between individual proton pairs. For nucleosides, the distance between the ribose protons H1′ and H2′ (r H1′-H2′ ) is 2.90 ( 0.2 Å regardless of the ribose conformation or the glycosyl torsional angle (28,38,39). The cross-relaxation rate between a pair of protons A and B (R A-B ) was, therefore, compared to the cross-relaxation rate of the H 1′ -H 2′ proton pair (R H1′-H2′ ), and the internuclear distance between protons A and B (r A-B ) was calculated using eq 1.…”

Section: Methodsmentioning

confidence: 99%

The Conformations of a Substrate and a Product Bound to the Active Site of S-Adenosylmethionine Synthetase

Schalk-Hihi

Markham

1999

Biochemistry

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S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.

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Section: Methodsmentioning

confidence: 99%

The Conformations of a Substrate and a Product Bound to the Active Site of S-Adenosylmethionine Synthetase

Schalk-Hihi

Markham

1999

Biochemistry

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“…19,24 As found by NMR with parallel kinetic assays, in solution the enzyme-bound ATP shows a high-antiglycosyl torsional angle ( ϭ 78 Ϯ 10°) when the enzyme is fully active, which decreases to a low-antiglycosyl torsional angle ( ϳ30°) as activity is lost. 25 The crystalline enzymes show low-antiglycosyl conformations for bound ATP ( ϭ 32°1 8 ; ϭ 47°1 9 ), suggesting that the enzyme may have lost some activity during the prolonged collection of X-ray data.…”

Section: Protein Kinasesmentioning

confidence: 99%

Mechanisms of signaling and related enzymes

Mildvan¹

1997

Proteins

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Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus. When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are. The mechanisms of cell signaling enzymes range from < or = 8% associative (cAMP-dependent protein kinase) to approximately 50% associative (G protein Gi alpha 1). Their catalytic powers range from 10(5.7) (p21ras) to 10(11.7) (lambda Ser-Thr protein phosphatase), usually comparable in magnitude with those of nonsignaling enzymes of the same mechanistic class. Exceptions are G proteins, which are 10(3)- to 10(5)-fold poorer catalysts than F1 and myosin ATPases. The lower catalytic powers of G proteins may be ascribed to the absence of general base catalysis, and additionally in the case of p21ras, to the absence of a catalytic Arg residue, which interacts with the transition state. From kinetic studies of mutant and metal ion substituted enzymes, the catalytic powers of cell signaling and related enzymes can be rationalized quantitatively by factors contributed by metal ion catalysis (> or = 10(5), general acid catalysis (approximately 10(3 +/- 1)), general base catalysis (approximately 10(3 +/- 1)), and transition-state stabilization by cationic and hydrogen bond donating residues (approximately 10(3 +/- 1)).

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“…Longitudinal relaxation rates ( l / T , ) were measured by a selective saturation-recovery method (Rosevear et al, 1983). …”

Section: Nmr Sample Conditions and Concentrationsmentioning

confidence: 99%

“…It is also clear from binding studies (Ferrin & Mildvan, 1985b) that dGTP occupies only one site per enzyme molecule under these conditions. The method of Rosevear et al (1983), using established conformations of deoxyguanosine as a basis set, was used to estimate the fractional contribution of each conformation to the observed average. The simplest fit to the average distances of Table IV was obtained by assuming a 60 f 5% contribution of the anti conformation found for dGTP in the presence of 01igo(rU)~~+~ as described above and a 40 f 5% contribution from a structure with a syn torsional angle (x = 222') and a C2'-endo deoxyribose pucker (6 = 144'), as has been found in the X-ray structure of deoxyguanosine (Haschemeyer & Sobell, 1965).…”

Section: Conformation Of Enzyme-bound Dgtp In the Presence And Absencmentioning

confidence: 99%

NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Ferrin¹,

Mildvan²

1986

Biochemistry

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The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)

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Nuclear Overhauser effect studies of the conformations of tetraamminecobalt(III)-ATP free and bound to bovine heart protein kinase

Cited by 46 publications

References 39 publications

The Conformations of a Substrate and a Product Bound to the Active Site of S-Adenosylmethionine Synthetase

The Conformations of a Substrate and a Product Bound to the Active Site of S-Adenosylmethionine Synthetase

Mechanisms of signaling and related enzymes

NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

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