Biomedical subjects

J G Clifton

Publications and source records attributed to J G Clifton.

4 recordsLinked to original sources

PubMed2001

THEMATICS: a simple computational predictor of enzyme function from structure.

We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in proteins of known structure. Results are featured for three enzymes: triosephosphate isomerase (TIM), aldose reductase (AR), and phosphomannose isomerase (PMI). We note that TIM and AR have similar structures but catalyze different kinds of reactions, whereas TIM and PMI have different structures but catalyze similar reactions. Analysis of the theoretical microscopic titration curves for all of the ionizable residues of these proteins shows that a small fraction (3-7%) of the curves possess a flat region where the residue is partially protonated over a wide pH range. The preponderance of residues with such perturbed curves occur in the active site. Additional results are given in summary form to show the success of the method for proteins with a variety of different chemistries and structures.

Aldehyde Reductase↗

PubMed1996

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N mutant.

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J.A., Gerlt, J.A., Kozarich, J.W., Koo, C.W., Shah, V.J., Kenyon, G.L., Neidhart, D.J., Fujita, J., & Petsko, G.A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R)- and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., Kenyon, G.L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.R., Petsko, G.A., & Kenyon, G.L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S)-atrolactate, with the exception that the side chain of His 297 is tilted and displaced approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S)-mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p-(bromomethyl)mandalate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pKa of His 297 in wild-type MR.

Base Sequence↗

PubMed1995

Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.

In the high-resolution X-ray structure of mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrogen-bonded to the carboxylate group of the bound inhibitor. This geometry suggests that the carboxylic acid functional group of Glu 317 participates as a general acid catalyst in the concerted general acid-general base catalyzed formation of a stabilized enolic tautomer of mandelic acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray structural analysis in the presence of (S)-atrolactate. No conformational alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for kcat were reduced 4.5 x 10(3)-fold for (R)-mandelate and 2.9 x 10(4)-fold for (S)-mandelate; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].

Base Sequence↗

PubMed1995

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the K166R mutant.

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts that either abstract the alpha-proton from the enantiomers of mandelate to form an enolic intermediate or protonate the enolic intermediate to form the enantiomers of mandelate, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. In this paper we report the structural and mechanistic properties of the mutant in which Lys 166 has been replaced with arginine (K166R). The structure of K166R has been determined at 1.85 A resolution with the substrate (S)-mandelate bound in the active site. The structure of this complex reveals no geometric alterations in the active site, with the exception that the longer side chain of Arg 166 is necessarily displaced upward from the position occupied by Lys 166 by steric interactions with the bound substrate. In contrast to the H297N mutant of MR [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9275-9281], the K166R exhibits low levels of racemase activity [kcat is reduced 5 x 10(3)-fold in the (R)- to (S)-direction and 1 x 10(3)-fold in the (S)- to (R)-direction]. The substrate and solvent deuterium isotope effects support a reaction coordinate for the K166R-catalyzed reaction in which the transition state for interconversion of bound (S)-mandelate and the stabilized enolic intermediate is higher in energy that the transition state for interconversion of bound (R)-mandelate and the stabilized enolic intermediate. The solvent deuterium isotope effect when (S)-mandelate is substrate (2.2 +/- 0.3) supports the proposal that the formation of the enolic intermediate involves partial transfer of a solvent-derived proton from Glu 317 to the substrate as the alpha-proton is abstracted [Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2777-2787].(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗