Transition State Structure of Purine Nucleoside Phosphorylase and Principles of Atomic Motion in Enzymatic Catalysis<sup>,</sup>

Fedorov, A.A.; Shi, Wuxian; Kicska, Greg A.; Fedorov, E.V.; Tyler, Peter C.; Furneaux, Richard H.; Hanson, John C.; Gainsford, Graeme J.; Larese, J. Z.; Schramm, Vern L.; Almo, Steven C.

doi:10.1021/bi002499f

Cited by 203 publications

(285 citation statements)

References 29 publications

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“…For this scheme, assuming the chemical step is irreversible (i.e., k 4 and reverse commitment, C r = 0), the V/K KIE, symbolized as T (V/K), is related to the equilibrium BIE ( T K eq ) and the intrinsic KIE ( T k 3 ) by Equation 6 (46,47): (6) where T k 1 is the intrinsic KIE for binding and C f is the forward commitment to catalysis, defined as k 3 /k 2 . In cases where C f is low, the T k 1 C f term in Equation 6 can be approximated within experimental error as C f , 4 yielding Equation 7:…”

Section: Intrinsic Kinetic Isotope Effects From V/k Kinetic Isotope Ementioning

confidence: 99%

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

et al. 2007

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The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition state analogues show His 257 within hydrogen-bonding distance to the 5′-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (K m /K i ) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His 257 serves an important role in the early stages of transition state formation. Whereas mutation of His 257 resulted in little variation in the PNP·DADMe-ImmH·SO 4 structures, His257Phe·ImmH·PO 4 showed distortion at the 5′-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies on the remote 5′-3 H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5′-3 H intrinsic KIE to −3%, −14%, and 7%, respectively, while the BIEs contributed 2%, 2%, and −2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state. Keywordspurine nucleoside phosphorylase; mutagenesis; binding isotope effect; kinetic isotope effect; transition state; binding distortion; transition state geometry; dynamics Determination of the transition state structure of enzymatic reactions allows for the design of tight binding transition state analogue inhibitors. These transition state mimics can subvert the energetics that govern formation of the enzymatic transition state to achieve binding orders of magnitude stronger than that of substrate. The transition state structures of enzymatic reactions have frequently been established by measuring kinetic isotope effects (KIEs 1 ) from the competitive reaction of isotopically labeled substrates. These isotope effects on k cat /K m , often † Supported by NIH Research Grant GM41916.* To whom correspondence should be addressed. vern@aecom.yu.edu; Telephone, (718) 430-2813; Fax, (718) . ‡ Current address: Department of Chemistry, Barnard College, 3009 Broadway, New York, NY 10027. § Current address: Center for Synchrotron Bioscience, Brookhaven National Laboratory, Upton, NY 11973.1 Abbreviations: KIE, kinetic isotope effect; V/K KIE, kinetic isotope effect on the second-order rate constant, k cat /K m ; BIE, binding isotope effect; HsPNP, human purine nucleoside phosphorylase; PDB, Protein Data Bank; ImmH, Immucillin-H; DADMe-ImmH, 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H; RMS, root mea...

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Section: Intrinsic Kinetic Isotope Effects From V/k Kinetic Isotope Ementioning

confidence: 99%

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

et al. 2007

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“…In particular, the functional role of fast picosecond protein motions in catalysis and the dynamic nature of the transition state barrier crossing is a subject of ongoing and current debate (7)(8)(9)(10)(11)(12)(13). Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22). In an alternative viewpoint, preorganization effects have been suggested to be a major contributing factor to enzyme catalysis (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Jha¹,

Gaffney

et al. 2011

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

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Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ 5 -3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.enzyme catalysis | electrostatic preorganization | Stark effect | visible-pump IR probe | time-resolved anisotropy E nzymes catalyze the vast majority of biochemical reactions and accelerate the rate of these reactions by many orders of magnitude compared to the uncatalyzed reactions in solution. The origins of the enormous catalytic power of enzymes are still not well understood despite enormous effort (1-6). In particular, the functional role of fast picosecond protein motions in catalysis and the dynamic nature of the transition state barrier crossing is a subject of ongoing and current debate (7-13). Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22). In an alternative viewpoint, preorganization effects have been suggested to be a major contributing factor to enzyme catalysis (23-28). It has been postulated that enzymes have partially oriented dipoles of polar and charged groups in the active site that interact with electrostatic features present in the catalytic transition state more favorably than water can. Such preorganization of active site dipoles and charges has been suggested to result in a reduction in the reorganization energy and provide enormous catalytic advantage over the reaction in solution, where water molecules must rearrange in order to solvate charge rearrangements during the chemical reaction (24,(29)(30)(31). The relative merits of these opposing viewpoints remains unclear largely because of the paucity of direct experimental assessment of the key discrepancies betwee...

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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 Purine Nucleoside Phosphorylase and Principles of Atomic Motion in Enzymatic Catalysis^,

Cited by 203 publications

References 29 publications

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

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

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Transition State Structure of Purine Nucleoside Phosphorylase and Principles of Atomic Motion in Enzymatic Catalysis,

Cited by 203 publications

References 29 publications

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

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

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Transition State Structure of Purine Nucleoside Phosphorylase and Principles of Atomic Motion in Enzymatic Catalysis^,