The Mechanism of Action of Aldolases

Morse, Daniel E.; Horecker, B.L.

doi:10.1002/9780470122761.ch4

Cited by 38 publications

(12 citation statements)

References 153 publications

(62 reference statements)

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“…Nonetheless, whether human aldolase A binds the substrate with positive homotropic cooperativity (which, for a tetramer has a potential range from 1 to 4) has not been well established. Positive cooperativity has been reported for aldolase A, but so has both negative cooperativity and an absence of cooperativity 12,15–20 . Enzyme inactivation studies with D‐glyceraldehyde‐phosphate and inorganic phosphate show that the modification of one subunit was sufficient to abolish aldolase activity, consistent with possible regulatory interactions among active sites 21 …”

Section: Resultsmentioning

confidence: 78%

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population

Fenton

Meneely

et al. 2021

Protein Science

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Some protein positions play special roles in determining the magnitude of protein function: at such “rheostat” positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A (“I98X”). Despite being ~17 Å from the active site and far from subunit interfaces, substitutions at position 98 have rheostatic contributions to the apparent cooperativity (nH) associated with fructose‐1,6‐bisphosphate substrate binding and moderately affected binding affinity. Next, we crystallized representative I98X variants to assess structural consequences. Residues smaller than the native isoleucine (cysteine and serine) were readily accommodated, and the larger phenylalanine caused only a slight separation of the two parallel helixes. However, the diffraction quality was reduced for I98F, and further reduced for I98Y. Intriguingly, the resolutions of the I98X structures correlated with their nH values. We propose that substitution effects on both nH and crystal lattice disruption arise from changes in the population of aldolase A conformations in solution. In combination with results computed for rheostat positions in other proteins, the results from this study suggest that rheostat positions accommodate a wide range of side chains and that structural consequences manifest as shifted ensemble populations and/or dynamics changes.

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Section: Resultsmentioning

confidence: 78%

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population

Fenton

Meneely

et al. 2021

Protein Science

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“…The K M of our ribozyme is in the same range as that of other biological aldol catalysts, such as the class I enzyme fructose-1,6-diphosphate-aldolase from mammals, with a K M of 0.3-1.0 mM (depending on the tissue [32]) for the natural substrate D-glycerinaldehyde-3-phosphate and dihydroxyacetone phosphate. Other catalysts for the aldol reaction, which utilize a mechanism common to natural class I aldolases, are the catalytic antibodies 38C2 and 33F12 [33,34], obtained through immunization in vivo.…”

Section: Kinetic Characterization Of the 11d2 Ribozymementioning

confidence: 71%

A Ribozyme for the Aldol Reaction

Fusz

Eisenführ

Srivatsan

et al. 2005

Chemistry & Biology

130

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Directed in vitro evolution can create RNA catalysts for a variety of organic reactions, supporting the "RNA world" hypothesis, which proposes that metabolic transformations in early life were catalyzed by RNA molecules rather than proteins. Among the most fundamental carbon-carbon bond-forming reactions in nature is the aldol reaction, mainly catalyzed by aldolases that utilize either an enamine mechanism (class I) or a Zn(2+) cofactor (class II). We report on isolation of a Zn(2+)-dependent ribozyme that catalyzes an aldol reaction at its own modified 5' end with a 4300-fold rate enhancement over the uncatalyzed background reaction. The ribozyme can also act as an intermolecular catalyst that transfers a biotinylated benzaldehyde derivative to the aldol donor substrate, coupled to an external hexameric RNA oligonucleotide, supporting the existence of RNA-originated biosynthetic pathways for metabolic sugar precursors and other biomolecules.

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“…In most enzymes catalysing such reactions, this step is facilitated through various means of stabilising the resulting enol(ate). For instance, class I aldolases use covalent catalysis involving a Schiff base mechanism (Gefflaut et al , 1995), whereas class II aldolases contain a metal ion to stabilise the enol(ate) intermediate (Morse and Horecker, 1968). The high‐resolution structure of SnoaL with bound product reveals that this polyketide cyclase does not use any of the above mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation

Sultana

Kallio

Jansson

et al. 2004

EMBO J

126

146

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SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 Å resolution. The fold of the subunit can be described as a distorted a þ b barrel, and the ligand is bound in the hydrophobic interior of the barrel. The 3D structure and site-directed mutagenesis experiments reveal that the mechanism of the intramolecular aldol condensation catalysed by SnoaL is different from that of the classical aldolases, which employ covalent Schiff base formation or a metal ion cofactor. The invariant residue Asp121 acts as an acid/base catalyst during the reaction. Stabilisation of the enol(ate) intermediate is mainly achieved by the delocalisation of the electron pair over the extended p system of the substrate. These polyketide cyclases thus form of family of enzymes with a unique catalytic strategy for aldol condensation.

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The Mechanism of Action of Aldolases

Cited by 38 publications

References 153 publications

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population

A Ribozyme for the Aldol Reaction

Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation

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