Leaving Group Activation and Pyrophosphate Ionic State at the Catalytic Site ofPlasmodium falciparumOrotate Phosphoribosyltransferase

Yong Zhang; Hua Deng; Vern L. Schramm

doi:10.1021/ja107806j

Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805416115 ◽

2018 ◽

Vol 115 (27) ◽

pp. E6209-E6216 ◽

Cited By ~ 12

Author(s):

Rajesh K. Harijan ◽

Ioanna Zoi ◽

Dimitri Antoniou ◽

Steven D. Schwartz ◽

Vern L. Schramm

Keyword(s):

Transition State ◽

Purine Nucleoside Phosphorylase ◽

Isotope Effects ◽

Catalytic Site ◽

Path Sampling ◽

Transition Path ◽

Transition Path Sampling ◽

Steady State Kinetics ◽

Leaving Group ◽

Transition State Barrier

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13C, 15N, and nonexchangeable 2H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly (kchemlight/kchemheavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster (kchemlight/kchemheavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchemlight/kchemheavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts.

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The Catalytic Mechanisms of the Reactions between Tryptophan Indole-Lyase and Nonstandard Substrates: The Role of the Ionic State of the Catalytic Group Accepting the Cα Proton of the Substrate

Acta Naturae ◽

10.32607/20758251-2019-11-3-82-88 ◽

2019 ◽

Vol 11 (3) ◽

pp. 82-88

Author(s):

N. G. Faleev ◽

M. A. Tsvetikova ◽

O. I. Gogoleva ◽

V. V. Kulikova ◽

S. V. Revtovich ◽

...

Keyword(s):

Acid Catalysis ◽

Isotope Effects ◽

Subsequent Stage ◽

Basic Form ◽

Ionic State ◽

Catalytic Function ◽

General Acid ◽

Leaving Group ◽

Mechanism Of Hydrolysis ◽

Solvent Isotope

In the reaction between tryptophan indole-lyase (TIL) and a substrate containing a bad leaving group (L-serine), general acid catalysis is required for the group's elimination. During this stage, the proton originally bound to the C atom of the substrate is transferred to the leaving group, which is eliminated as a water molecule. As a result, the basic group that had accepted the C proton at the previous stage has to be involved in the catalytic stage following the elimination in its basic form. On the other hand, when the substrate contains a good leaving group (-chloro-L-alanine), general acid catalysis is not needed at the elimination stage and cannot be implemented, because there are no functional groups in enzymes whose acidity is strong enough to protonate the elimination of a base as weak as Cl- anion. Consequently, the group that had accepted the C proton does not lose its additional proton during the elimination stage and should take part in the subsequent stage in its acidic (not basic) form. To shed light on the mechanistic consequences of the changes in the ionic state of this group, we have considered the pH dependencies of the main kinetic parameters for the reactions of TIL with L-serine and -chloro-L-alanine and the kinetic isotope effects brought about by replacement of the ordinary water used as a solvent with 2H2O. We have found that in the reaction between TIL and -chloro-L-alanine, the aminoacrylate hydrolysis stage is sensitive to the solvent isotope effect, while in the reaction with L-serine it is not. We have concluded that in the first reaction, the functional group containing an additional proton fulfills a definite catalytic function, whereas in the reaction with L-serine, when the additional proton is absent, the mechanism of hydrolysis of the aminoacrylate intermediate should be fundamentally different. Possible mechanisms were considered.

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Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

Biochemical Journal ◽

10.1042/bj20031966 ◽

2004 ◽

Vol 381 (1) ◽

pp. 125-130 ◽

Cited By ~ 7

Author(s):

Sanjiv SONKARIA ◽

Guillaume BOUCHER ◽

José FLÓREZ-ÁLVAREZ ◽

Bilal SAID ◽

Syeed HUSSAIN ◽

...

Keyword(s):

Hydrolytic Enzymes ◽

Catalytic Site ◽

Catalytic Antibody ◽

Substrate Selectivity ◽

Reaction Centre ◽

Antibody Preparation ◽

Leaving Group ◽

Catalytically Active ◽

Group Part ◽

Kinetic Selectivity

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.

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Dual-Function, Light Switchable Cobalt Catalysis via Active Site Metamorphosis

10.26434/chemrxiv.11353106.v1 ◽

2019 ◽

Author(s):

Enrico Bergamaschi ◽

Frédéric Beltran ◽

Christopher Teskey

Keyword(s):

Visible Light ◽

Active Site ◽

Catalytic Site ◽

Dual Function ◽

Catalytic Systems ◽

Hydride Complex ◽

Dual Functional ◽

Multiple Processes ◽

Olefin Migration

Switchable catalysis offers opportunities to control the rate or selectivity of a reaction via a stimulus such as pH or light. However, few examples of switchable catalytic systems that can facilitate multiple processes exist. Here we report a rare example of such dual-functional, switchable catalysis. Featuring an easily prepared, bench-stable cobalt(I) hydride complex in conjunction with pinacolborane, we can completely alter the reaction outcome between two widely employed transformations – olefin migration and hydroboration – with visible light as the sole trigger. This dichotomy arises from ligand photodissociation which leads to metamorphosis of the active catalytic site, resulting in divergent mechanistic pathways.

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General Cyclopropane Assembly via Enantioselective Redox-Active Carbene Transfer to Aliphatic Olefins

10.26434/chemrxiv.7436795 ◽

2018 ◽

Author(s):

Marc Montesinos-Magraner ◽

Matteo Costantini ◽

Rodrigo Ramirez-Contreras ◽

Michael E. Muratore ◽

Magnus J. Johansson ◽

...

Keyword(s):

Total Synthesis ◽

Asymmetric Synthesis ◽

Chemical Space ◽

Synthetic Approach ◽

Asymmetric Cyclopropanation ◽

Redox Active ◽

Wide Range ◽

Leaving Group ◽

Stereoelectronic Properties ◽

Carbene Transfer

Asymmetric cyclopropane synthesis currently requires bespoke strategies, methods, substrates and reagents, even when targeting similar compounds. This limits the speed and chemical space available for discovery campaigns. Here we introduce a practical and versatile diazocompound, and we demonstrate its performance in the first unified asymmetric synthesis of functionalized cyclopropanes. We found that the redox-active leaving group in this reagent enhances the reactivity and selectivity of geminal carbene transfer. This effect enabled the asymmetric cyclopropanation of a wide range of olefins including unactivated aliphatic alkenes, enabling the 3-step total synthesis of (–)-dictyopterene A. This unified synthetic approach delivers high enantioselectivities that are independent of the stereoelectronic properties of the functional groups transferred. Our results demonstrate that orthogonally-differentiated diazocompounds are viable and advantageous equivalents of single-carbon chirons.

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