His···Asp Catalytic Dyad of Ribonuclease A:  Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Schultz, L. Wayne; Quirk, David J.; Raines, Ronald T.

doi:10.1021/bi972766q

Cited by 77 publications

(106 citation statements)

References 87 publications

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“…The role of these two histidine residues in the conformational exchange process in RNase A was explored further. H119 makes an important H bond with the ␥-carboxyl of D121, which serves to connect H119 to loop 4 and the main body of the protein (19,30). H119 is not located in a region of the enzyme involved in the isotope-sensitive motion.…”

Section: Resultsmentioning

confidence: 99%

The mechanism of rate-limiting motions in enzyme function

Watt

Shimada

Kovrigin

et al. 2007

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

145

201

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The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of Ϸ160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.Carr-Purcell-Meiboom-Gill dispersion ͉ enzyme dynamics ͉ NMR ͉ protein motions ͉ RNase A C onformational motions in enzymes play an essential role in their function and are often the rate-limiting step to overall catalytic throughput (1-5). Many enzymes have sufficiently evolved such that the bond-making and -breaking steps are fast relative to the ability of the enzyme to undergo a conformational change, and, thus, steps other than the chemical transformation of substrate are rate-limiting (ref. 6 and references therein). In systems such as these, understanding enzyme function requires characterization of the relevant time-dependent protein fluctuations from the timeaveraged three-dimensional structure. The ability of solution NMR spectroscopy to detect, with atomic resolution, motions over a wide timescale (picoseconds to seconds) makes it an ideal experimental technique to characterize conformational motions in proteins that can ultimately impact drug design, de novo enzyme construction, and enzyme engineering. In particular, relaxation-compensated Carr-Purcell-Meiboom-Gill (rcCPMG) dispersion experiments (7) are capable of informing on the kinetics, thermodynamics, and structural changes of protein motions in the microsecond-tomillisecond timescale.RNase A is an enzyme example in which a conformational change is the bottleneck to overall conversion of substrate to product (see ref. 3 for a review). RNase A catalyzes the cleavage of single-stranded RNA and does not require metal ions or cofactors. This enzyme has been studied in great detail as a model for protein folding, structure, and stability (8, 9). In addition, homologs of RNase A have important cytotoxic and antitumor properties (10). The rate-limiting step for the RNase A reaction is a protein conformational change that is coupled to the product release step (1, 11). This conformational change involves multiple amino acid residues throughout the protein, including those distant from the active site (12-14). These mobile regions include two loops: loop 1,...

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Section: Resultsmentioning

confidence: 99%

The mechanism of rate-limiting motions in enzyme function

Watt

Shimada

Kovrigin

et al. 2007

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

145

201

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“…This structural motif, which further restricts the conformational space available to the Glu residue, is not found in ZF-RNase-1, where the Asp residue is replaced by a His. In this respect, it should be recalled that the corresponding aspartate of RNase A (Asp121) forms a sort of "catalytic dyad" with His119 [30]. In the bovine pancreatic enzyme, the H-bond between the side chains of these two residues stabilizes the histidine conformation that is supposed to be active during the transphosphorylation step.…”

Section: Discussionmentioning

confidence: 99%

“…In ZF-RNase-5 this helix, which encompasses residues [24][25][26][27][28][29][30][31][32][33][34][35], has a first turn in a 3 10 conformation, and terminates with a turn in a π helix conformation with main chain hydrogen bonds Met31-Ile36 and Ser32-Lys35 and Lys35 in the Lα conformation. In the corresponding region (residues 23-36) of ZF-RNase-1 [4] the network of hydrogen bonds (Ile30-Ile37 and Gly31-Lys36) is spatially conserved.…”

Section: Introductionmentioning

confidence: 99%

“…Thus the topology of this fragment is substantially unchanged with respect to ZF-RNase-5, although the insertion of the two residues formally breaks the helix at the level of Ile30 (Figure 2 and supplemental Figure S3). In the case of ZF-RNase-3 (residues [23][24][25][26][27][28][29][30][31][32][33][34][35] [4] the first turn of the helix is highly distorted.…”

Section: Introductionmentioning

confidence: 99%

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A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

Pizzo

Merlino

Turano

et al. 2010

Biochemical Journal

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Recently, extracellular RNases of the RNase A superfamily, with the characteristic CKxxNTF sequence signature, have been identified in fish. This has led to the recognition that these RNases are present in the whole vertebrate subphylum. In fact, they comprise the only enzyme family unique to vertebrates. Four RNases from zebrafish (Danio rerio) have been previously reported and have a very low RNase activity; some of these are endowed, like human angiogenin, with powerful angiogenic and bactericidal activities. In the present paper, we report the three-dimensional structure, the thermodynamic behaviour and the biological properties of a novel zebrafish RNase, ZF-RNase-5. The investigation of its structural and functional properties, extended to all other subfamily members, provides an inclusive description of the whole zebrafish RNase subfamily

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“…We compare the structure of P93G RNase A to that of crystalline D121ARNaseA (pdb entry 4rsd; Schultz et al, 1998). The D121A variant has a structure that is almost identical (0.37 8, RMSD of the main chain) to that of the crystalline wild-type protein in a concentrated salt solution (pdb entry lrph).…”

mentioning

confidence: 99%

Structure and stability of the P93G variant of ribonuclease A

et al. 1998

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Abstract:The peptide bonds preceding Pro 93 and Pro I14 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 w.This structure is essentially identical to that of the wild-type protein, except for the 9 1-94 p-turn containing the substitution. In the wild-type protein, the p-turn is of type Vla. In the P93G variant, this turn is of type I1 with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of AAGm, which reports on the stability lost in the variants, is 1 S-fold greater for the PI 14G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type I1 turn, which has a preference for a glycine residue in its i + 2 position.

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His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Cited by 77 publications

References 87 publications

The mechanism of rate-limiting motions in enzyme function

The mechanism of rate-limiting motions in enzyme function

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

Structure and stability of the P93G variant of ribonuclease A

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