Allosteric Interactions within Subsites of a Monomeric Enzyme: Kinetics of Fluorogenic Substrates of PI-Specific Phospholipase C

Birrell, G. Bruce; Zaikova, Tatiana; Rukavishnikov, Aleksey V.; Keana, John F. W.; Griffith, O. Hayes

doi:10.1016/s0006-3495(03)70051-4

Cited by 24 publications

(21 citation statements)

References 36 publications

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“…Occupancy of both catalytic and regulatory sites within the overall extended peptide binding site is thus an important determinant for efficient trimming. Similar subsite activation models have been proposed to explain the length preference of bovine RNAse A43, and the sigmoidal velocity plots observed for the monomeric B. subtilis PI-PLC when assayed with short-chain lipid analog substrates44. The location of the regulatory site(s) within the ERAP1 structure is not yet clear in the absence of structural data for a bound peptide complex, but one possible site might be the inner surface of top of helices H20 and H22, which in the closed conformation would contact an extended version of the tripeptide model shown in Fig.…”

Section: Discussionsupporting

confidence: 62%

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

Nguyen

Chang

Evnouchidou

et al. 2011

Nat Struct Mol Biol

192

362

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ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

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Section: Discussionsupporting

confidence: 62%

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

Nguyen

Chang

Evnouchidou

et al. 2011

Nat Struct Mol Biol

192

362

View full text Add to dashboard Cite

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“…PC surfaces enhance cIP hydrolysis, whereas anionic surfaces inhibit enzyme activity (by acting as competitive inhibitors). Similar effects are also observed for the phosphotransferase activity of PI-PLC using long-chain and shortchain PI (6, 12) as well as monomeric nonlipid substrates (9). It has been documented that several peripheral membrane proteins oligomerize when bound to membranes (e.g.…”

Section: Discussionsupporting

confidence: 55%

“…For the soluble cIP reaction, k cat was increased, and K m decreased in the presence of PC interfaces (4,8). That work suggested that PC binds to an activator site on the bacterial enzyme that is distinct from the enzyme active site, a model confirmed recently using artificial substrates (9). However, the details of the conformational change upon PC binding that leads to improved enzyme efficiency are not known.…”

mentioning

confidence: 85%

Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme

Zhang

Wehbi

Roberts

2004

Journal of Biological Chemistry

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Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC 7 PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC 7 PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this crosslinking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (␤␣) 8 -barrel protein. The higher activity of the native protein cross-linked in the presence of diC 7 PC correlated with an increased affinity of the protein for two diC 7 PC molecules as detected by matrixassisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC 7 PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

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“…Enzymes that display a positive cooperativity with a Hill coefficient higher than 1 do not always correspond to an oligomeric structure. It is well established that there is a class of monomeric enzymes that display positive cooperativity for substrate binding (41)(42)(43)(44)(45). This positive cooperativity has been explained by a theoretical "mnemonical" model (43).…”

Section: Figmentioning

confidence: 99%

The Escherichia coli RecQ Helicase Functions as a Monomer

Xu¹,

Deprez²,

Zhang³

et al. 2003

Journal of Biological Chemistry

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The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 M and in a temperature range between 4 and 37°C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5-adenylyl-␤,␥-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and singlestranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.Genetic information is stored into the double-stranded DNA molecule that is stabilized through specific hydrogen-bonded base pairs. However, replication and repair as well as recombination of the DNA molecule require a single-stranded DNA to be available at least transiently. In cells, unwinding and separation of the complementary strands of duplex DNAs (dsDNA) 1 into single-stranded DNAs (ssDNA) are catalyzed by a class of enzymes known as helicases. DNA helicases destabilize and unwind the duplex DNA through a series of energetic states, driven by the binding and hydrolysis of NTP, usually ATP, and subsequent release of NDP and inorganic phosphate. Thus, DNA helicases convert the chemical energy into mechanical energy for DNA unwinding and translocation along the nucleic acid lattice (for reviews, see Refs. 1-3).During unwinding of dsDNA, and to translocate processively without dissociation from DNA, the helicase must use at least two DNA-binding sites to keep contact with the DNA lattice; one binds to ssDNA for translocating, whereas the other binds to dsDNA for DNA unwinding. These two DNA binding sites may be located at different domains within a single polypeptide of a monomer or be held by two different polypeptides within a dimer or an oligomer for providing multiple DNA-binding sites. Corresponding to the first possibility, the "inchworm" model was proposed, in which the helicase is assumed to possess two nonidentical DNA binding sites (4); the "leading" site binds both ssDNA and dsDNA and interacts with the duplex to be unwound during s...

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Allosteric Interactions within Subsites of a Monomeric Enzyme: Kinetics of Fluorogenic Substrates of PI-Specific Phospholipase C

Cited by 24 publications

References 36 publications

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme

The Escherichia coli RecQ Helicase Functions as a Monomer

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