The Active Sites of Fructose 6-Phosphate,2-kinase: Fructose-2,6-bisphosphatase from Rat Testis

Uyeda, Kosaku; Wang, Xiao Li; Mizuguchi, Hiroyuki; Li, Yang; Nguyen, Cu Le; Hasemann, Charles A.

doi:10.1074/jbc.272.12.7867

Cited by 17 publications

(28 citation statements)

References 38 publications

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“…For competition assays, 0.4 M motors were incubated with 2 M Mt and 2 M mADP and titrated against varying concentrations of unlabeled ADP. The IC 50 , defined as the [ADP] where the fluorescence signal drops to 50%, was fit using Morrison's quadratic equation (31),…”

Section: Methodsmentioning

confidence: 99%

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Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Chen

Arginteanu

Hancock

2015

Journal of Biological Chemistry

115

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Background: KIF3A/B is less processive than kinesin-1, particularly under load. Results: In an engineered KIF3A dimer, intramolecular strain in the two-head-bound state does not alter nucleotide binding. In ADP, the motor dissociates slowly from microtubules. Conclusion: KIF3A lacks front head gating and maintains processivity through weak microtubule binding. Significance: During bidirectional transport, kinesin-2 is expected to detach more readily than kinesin-1.

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Section: Methodsmentioning

confidence: 99%

“…From the IC 50 , the relationship between the equilibrium binding constants for mADP and ADP was obtained using the Cheng-Prusoff equation (32),…”

Section: Methodsmentioning

confidence: 99%

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Chen

Arginteanu

Hancock

2015

Journal of Biological Chemistry

115

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“…A function in liganding to M values increase by Ϸ20-fold for ATP and Ϸ40-fold for mevalonate. Effects of this magnitude have been observed upon replacement of carboxyl groups that function as cation ligands in phosphofructokinase (25), 6-phosphofructo-2-kinase (22), and phosphoribulokinase (26). If the assignment of Glu-193 as cation ligand is correct, then, in the absence of any other invariant acidic residues that can be clearly be implicated in catalysis, a serine/threonine residue would be a plausible candidate for the role of second cation ligand.…”

Section: An Active Site Serine In Mevalonate Kinasementioning

confidence: 99%

“…Another potential active site role for alcohol functionalities in phosphotransferase reactions involves H-bonding to oxygens of the phosphoryl chain of ATP substrate (18 -20). Elimination of the side chain in such enzymes can produce a k cat effect (21,22) even larger than typically generated upon elimination of a cation ligand. Alignment of available deduced sequences for eukaryotic and prokaryotic mevalonate kinases indicates only four invariant residues (Ser-145, Ser-146, Ser-201, Thr-243) with alcohol groups in their side chains (Fig.…”

Section: Mutagenic Substitution Of Alcohol Sidementioning

confidence: 99%

Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

Cho

Rios

Kim

et al. 2001

Journal of Biological Chemistry

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Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in K m(ATP) . K m for mevalonate increases by Ϸ20-fold for S146A, Ϸ40-fold for T243A, and 100-fold for S201A. V max changes for S145A, S201A, and T243A are <3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V max for S146A is diminished by 4000-fold. In terms of V/K MVA , this substitution produces a 10 5 -fold effect, suggesting an active site location and catalytic role for Ser-146. The large k cat effect suggests that Ser-146 productively orients ATP during catalysis. K D(Mg-ATP) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg 2؉ -ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.Isoprenoid biosynthesis is accomplished by diverse pathways that use either acetyl-CoA (1) or glyceraldehyde 3-phosphate and pyruvate (2) as starting materials. Mevalonate kinase (E.C. 2.7.1.36) represents a distinctive component of the former pathway, which functions in eukaryotes, archaebacteria, and some eubacteria. In the mevalonate-mediated pathway for isoprenoid production, attention has long been focused on HMGCoA 1 reductase, which catalyzes a highly regulated step. The diversity of species in which mevalonate kinase functions is reflected in the high degree of heterology that is apparent upon comparison of deduced sequences for this enzyme. Only 5% of the amino acids that encode the human enzyme are invariant. Included in this select group are the residues (Lys-13, 2 Glu-193, Asp-204) that our laboratory has implicated previously (6, 7) in various active site functions. Inspection of mevalonate kinase's amino acid sequence does not suggest that substrate ATP binding relies on well established consensus sequences such as Walker A or Walker B motifs. However, two distinct glycine-rich stretches of amino acid sequence have been proposed as potential ATP-binding regions (8, 9), although no direct evidence that tests these hypotheses or discriminates between the two candidate sequences has appeared.For phosphotransferase enzymes, there are numerous examples of serines or threonines that map to the...

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“…Previously, we cloned a gene specifically expressed in human testis [8] that corresponds to the pfkfb4 gene [7] and encodes the testes PFK-2 isozyme [9,10]. Recently, it has been demonstrated that pfkfb4 gene has a strong response to hypoxic stimulation [11].…”

Section: Introductionmentioning

confidence: 99%

Specific expression of pfkfb4 gene in spermatogonia germ cells and analysis of its 5′‐flanking region

et al. 2004

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The results presented demonstrate the expression of pfkfb4 gene in adult testis and in a mouse spermatogonia germ cell line (GC-1spg). The genomic organization of the human pfkfb4 gene shows the existence of 14 exons and 13 introns, spanning 45 kb. A detailed analysis of the 5 0 -flanking region by transient transfection assays with different 5 0 -deletion promoter constructs in GC-1spg and mouse sertoli cells (TM-4), allows us to define the minimal promoter unit, containing several GC-rich and ETF sequences along the first À141 nucleotides involved in basal expression. This gene is activated by serum and chemical hypoxia (CoCl 2 treatment) whereas b-estradiol decreases its expression.

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The Active Sites of Fructose 6-Phosphate,2-kinase: Fructose-2,6-bisphosphatase from Rat Testis

Abstract: To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase (

Cited by 17 publications

References 38 publications

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

Specific expression of pfkfb4 gene in spermatogonia germ cells and analysis of its 5′‐flanking region

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