Profound contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: Mutation and rescue

Carlow, Dean C.; Smith, Albert A.; Yang, Charles C.; Short, Steven A.; Wolfenden, Richard

doi:10.1021/bi00013a010

Cited by 52 publications

(67 citation statements)

References 16 publications

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“…Protonation of a Zn 2ϩ -coordinating histidine is likely to destabilize the protein structure, and this is probably the reason for the decline of catalytic activity observed at pH 4.5. E. coli cytidine deaminase, which has a Zn 2ϩ -coordinating histidine, did not show a maximum reaction speed at pH 5.5 to 6.0 (31). Therefore, H257 is not likely the cause of the observed pH dependency of A3Gctd deamination.…”

Section: Discussionmentioning

confidence: 90%

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

Harjes

Solomon

et al. 2013

J Virol

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APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd's deamination reactions within a 5=-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5=-CCC substrate over 5=-CCU substrate, which explains why A3G displays almost no processivity within a 5=-CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5=-CCC flanked on both sides by a single nucleotide. A3Gctd as well as fulllength A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo. Human APOBEC3G (A3G) is a member of a family of Zn 2ϩ -dependent polynucleotide cytosine deaminases. This family was named after APOBEC1 (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1) and also includes the antibody gene diversification enzyme AID (activation-induced cytidine deaminase) (reviewed in references 1-5). A3G can restrict HIV-1 replication by packaging into assembling viral particles for delivery to target cells, where it deaminates cytosine to uracil in newly transcribed viral DNA. These cDNA uracils base pair with adenine during plus-strand synthesis and result in G-to-A hypermutation and, in turn, inactivation of the viral genome. A3G has two Zn 2ϩ binding domains that span residues 1 to 196 and 197 to 384, but only the C-terminal domain is catalytically active (6-8). The Nterminal domain interacts with HIV-1 Vif, RNA, and singlestranded DNA (ssDNA) (e.g., see references 7 and 9-11). A3G predominantly deaminates the 3= cytosine (underlined) in a 5=-CCC sequence, although the middle cytosine can also be deaminated in subsequent reactions following deamination of the 3= cytosine (12-16). In longer ssDNA substrates with multiple 5=-CCC sites, A3G deamination exhibits a 3=¡5= spatial preference in vitro (9,17,18). In the present study, we use the catalytic domain of A3G (A3Gctd) to determine kinetic parameters. Our results provide kinetic constants for two independent deaminations within a 5=-CCC sequence, which explain A3G's catalytic site preference for the 3= cytosine. We identify a strong pH dependence of the reaction speed, which implies that a histidine residue is involved in substrate binding. In addition, we identify the shortestlength ssDNA substrate for A3Gctd to be a pentanucleotide. MATERIALS AND METHODSPurification of A3Gctd. The APOBEC3G C-terminal domain (A3Gctd), comprising amino acids 191 to 384, was expressed and purified as previously described (19). Briefly, the glutathione S-transferase (GST)-fused A3Gctd was...

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Section: Discussionmentioning

confidence: 90%

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

Harjes

Solomon

et al. 2013

J Virol

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“…The fourth ligand position of the zinc ion is occupied by the catalytic water molecule (7). The second zinc solvation shell contains a glutamate residue (Glu104) that participates in the required proton transfers (7,8). The enzyme-substrate (ES) complex is in rapid equilibrium with free enzyme and substrate, and k cat solely represents the chemical steps, with the reaction being pH-independent in the neutral pH region (4).…”

Section: Significancementioning

confidence: 99%

Enzyme catalysis by entropy without Circe effect

Kazemi

Himo

Åqvist

2016

Proc. Natl. Acad. Sci. U.S.A.

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Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction in water, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.cytidine deaminase | density functional theory | empirical valence bond method | computational Arrhenius plots M any hypotheses have been put forward to explain the rate acceleration of chemical reactions by enzymes. One of the most influential of these is Jencks' so-called "Circe effect" (1), which posits that the key catalytic effect is associated with substrate binding and that part of the favorable (negative) binding free energy is spent on destabilization of the bound substrate in its ground state. Such ground-state destabilization could, in principle, have different possible physical origins, such as reduction of translational, rotational, and conformational substrate entropies; steric and conformational strain; or electrostatic destabilization and desolvation effects (1). In particular, the entropic explanation enjoys widespread popularity and is often invoked to rationalize the catalytic power of enzymes in terms of proximity and alignment of the reacting groups (2). It is then assumed that part of the substrate-binding free energy is spent on restricting the substrate motions and correctly aligning the substrate for reaction, which implies a negative contribution to the binding entropy. This scenario, in turn, would enable the substrate to climb the activation barrier with a smaller entropy loss than in solution, because the entr...

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“…Considerable effort has been devoted to study the catalytic mechanisms of the nucleoside deaminases cytidine deaminase (CDA) and adenosine deaminase [1][2][3][4][5][6][7][8][9][10][11][12][13]. The enzyme CDA is an efficient catalyst which accelerates the rate of hydrolytic deamination of cytidine to uridine.…”

Section: Introductionmentioning

confidence: 99%