Transition State Structure of <i>Salmonella typhimurium</i> Orotate Phosphoribosyltransferase

Tao, Wen; Grubmeyer, Charles; Blanchard, John S.

doi:10.1021/bi951898l

Cited by 90 publications

(135 citation statements)

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“…Members of one of the ribozyme families (family A) promoted the pyrimidine nucleotide synthesis reaction at least 10 6 times faster than the upper limit of the analogous uncatalyzed reaction (Unrau and Bartel 1998). Moreover, nucleotide synthesis and hydrolysis reactions, when promoted by protein enzymes, involve intermediates with considerable oxocarbenium character (Tao et al 1996;Chen et al 2000). This type of dissociative mechanism differs from the concerted reaction mechanisms that RNA is known to promote (Bartel and Unrau 1999), making the structure and mechanism of this ribozyme family of considerable interest.…”

Section: Introductionmentioning

confidence: 99%

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

Chapple

Bartel²,

Unrau³

2003

RNA

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We previously isolated from random sequences ribozymes able to form a glycosidic linkage between a ribose sugar and 4-thiouracil in a reaction that mimics protein-catalyzed nucleotide synthesis. Here we report on two serial in vitro selection experiments that defined the core motif of one of the nucleotide synthase ribozymes and provided improved versions of this ribozyme. The first selection experiment started from a degenerate sequence pool based on the previously isolated sequence and used a selection-amplification protocol that allowed the sequence requirements at the 3 terminus of the ribozyme to be interrogated. Comparing the active sequences identified in this experiment revealed the complicated secondary structure of the nucleotide synthase ribozyme. A second selection was then performed to remove nonessential sequence from the ribozyme. This selection started with a pool with variation introduced in both the sequence and the length of the nonconserved loops and joining regions. This pool was generated using a partial reblocking/deblocking strategy on a DNA synthesizer, allowing the combinatorial synthesis of both point deletions and point substitutions. The consensus ribozyme motif that emerged was an ∼71 nt pseudoknot structure with five stems and two important joining segments. Comparative sequence analysis and a cross-linking experiment point to the probable location of nucleotide synthesis. The prototype isolate from the second selection was nearly 35 times more efficient than the initial isolate and at least 10 8 times more efficient than an upper limit of an as-yet undetectable uncatalyzed reaction, supporting the idea that RNA-catalyzed nucleotide synthesis might have been important in an RNA world.

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Section: Introductionmentioning

confidence: 99%

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

Chapple

Bartel²,

Unrau³

2003

RNA

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“…However, the use of phosphonoacetic acid as a pyrophosphate analog made it possible to study the conversion of orotidine 5 -phosphate to orotate and 5-phosphoribosyl-1-phosphonoacetic acid under conditions that gave intrinsic kinetic isotope effects (100). In reactions with a concerted chemical step, the transition state structure can be determined from kinetic isotope effect measurements in either the forward or reverse directions because the same transition state defines both reactions.…”

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confidence: 99%

Enzymatic Transition States and Transition State Analog Design

Schramm

1998

Annu. Rev. Biochem.

273

264

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All chemical transformations pass through an unstable structure called the transition state, which is poised between the chemical structures of the substrates and products. The transition states for chemical reactions are proposed to have lifetimes near 10 −13 sec, the time for a single bond vibration. No physical or spectroscopic method is available to directly observe the structure of the transition state for enzymatic reactions. Yet transition state structure is central to understanding catalysis, because enzymes function by lowering activation energy. An accepted view of enzymatic catalysis is tight binding to the unstable transition state structure. Transition state mimics bind tightly to enzymes by capturing a fraction of the binding energy for the transition state species. The identification of numerous transition state inhibitors supports the transition state stabilization hypothesis for enzymatic catalysis. Advances in methods for measuring and interpreting kinetic isotope effects and advances in computational chemistry have provided an experimental route to understand transition state structure. Systematic analysis of intrinsic kinetic isotope effects provides geometric and electronic structure for enzyme-bound transition states. This information has been used to compare transition states for chemical and enzymatic reactions; determine whether enzymatic activators alter transition state structure; design transition state inhibitors; and provide the basis for predicting the affinity of enzymatic inhibitors. Enzymatic transition states provide an understanding of catalysis and permit the design of transition state inhibitors. This article reviews transition state theory for enzymatic reactions. Selected examples of enzymatic transition states are compared to the respective transition state inhibitors.

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“…Detailed analysis of the transition states for nucleoside phosphorylase (41) and OPRT (42) suggest that an important feature of the PRT mechanism is the concept of "nucleophillic displacement" (6). For nucleoside phosphorylase, this involves relatively fixed interactions to the nucleobase leaving group and Pi nucleophile, but a clear migration of C1′ (on the order 3 Å) from the leaving group to the Pi nucleophile.…”

Section: Implications For the Mechanism Of The Atp-prt Reactionmentioning

confidence: 99%

Substrate Recognition by the Hetero-Octameric ATP Phosphoribosyltransferase from Lactococcus lactis

2006

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Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms, and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisG S catalytic subunits related to periplasmic binding proteins, and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs, and among phosphoribosyltransferases in general, we determined the steady state kinetics for the heterooctameric form, and characterized the active site by mutagenesis. The K m PRPP (18.4 ± 3.5 μM) and k cat (2.7 ± 0.3 sec −1 ) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the K m for ATP (2.7 ± 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be non-competitive (K i = 81.1 μM) and competitive (K i = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments investigating the side chains recognizing PRPP showed that 5′ phosphate contacts (T159A and T162A) had the largest (25-and 155-fold) decreases in k cat /K m , while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety, or contacts to the 2′ OH. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters, and employ mechanistic strategies reminiscent of the broader PRT superfamily.ATP phosphoribosyltransferase (ATP-PRT; E. C. 2.4.2.17) catalyzes the first and highly regulated step of histidine biosynthesis, which involves nucleophillic attack of the N1 of ATP on C1′ of 5-phosphoribosyl-1 pyrophosphate (PRPP) to form N-1-(5′-phosphoribosyl)-ATP (PR-ATP, Figure 1A) (1). The resulting product is converted through an additional nine reactions into histidine by a pathway that is regulated both by its end product and by AMP and ADP (2). The tight regulation of this pathway (reviewed in (3)), reflects the high energetic costs associated with histidine synthesis, and the need to dramatically slow pathway flux when histidine is present in the growth media (4). Superimposed on top of metabolic control of † This work was supported by N.I.H. grant GM54899 (CSF) histidine biosynthesis is genetic regulation of the expression of histidine biosynthetic enzymes, by use of the classic attenuation mechanism (reviewed in (5)).ATP-PRT is a member of the phophoribosyltransferase (PRT) superfamily of enzymes, all of whom share a common chemistry that involves transfer of the phosphoribosyl group to a nucleotide base or, in the case of glutamine phosphoribosyl pyrophosphate amidotransferase, to free ammonia generated on the enzyme (6). PRTs are found in many essential pathways, including biosynthesis and salvage of nucleotides, and the synthesis of cofac...

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Transition State Structure of Salmonella typhimurium Orotate Phosphoribosyltransferase

Cited by 90 publications

References 30 publications

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

Enzymatic Transition States and Transition State Analog Design

Substrate Recognition by the Hetero-Octameric ATP Phosphoribosyltransferase from Lactococcus lactis

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