N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme.

N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococcus vannielii PR-AMP cyclohydrolase has been developed, and the first characterization of a purified form of the enzyme is reported. The enzyme has a native molecular weight of 31 200 as determined by analytical ultracentrifugation that agrees with the molecular mass determined by gel filtration (34 kDa) and a subunit molecular weight of 15 486 based on MALDI-MS. An unusual characteristic of the protein is the complexity observed on SDS-PAGE, and N-terminal amino acid sequence analysis of all the isolated constituents confirms their origin as PR-AMP cyclohydrolase. A highly conserved region of the amino acid sequence is implicated in the self-cleavage events of the protein and provides an explanation for the complexity of this protein. Bound to the enzyme is 1 equiv of Zn2+ that can be removed only by extended dialysis with 1,10-phenanthroline (Kd

Enoyl-coenzyme A hydratase-catalyzed exchange of the alpha-protons of coenzyme A thiol esters: a model for an enolized intermediate in the enzyme-catalyzed elimination?

3-Quinuclidinone catalyzes the exchange of the alpha-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10(-6) M-1 s-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10(2) M-1 s-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pKa of 8.8 and below a pKa of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10(5) as no incorporation of 2H into the alpha-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the alpha-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the alpha-protons becomes nonstereospecific when the acidity of the alpha-protons is enhanced. While alpha-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.

Electronic rearrangement induced by substrate analog binding to the enoyl-CoA hydratase active site: evidence for substrate activation.

A series of alpha,beta unsaturated CoA thiol esters have been characterized spectroscopically when they form noncovalent complexes at the active site of enoyl-CoA hydratase. The UV spectra of all of the thiol esters display significant red shifts when the esters are bound to the crotonase active site. The red shift increases with the ability of a para substituent of substituted cinnamoyl-CoA thiol esters to donate electrons by resonance. The affinity of the substituted cinnamoyl-CoA thiol esters is enhanced by electron-donating substituents, with the slope of the log of the ratio of the inhibition constants versus sigma p+ being near unity. Affinity is also increased by either para or meta electron-withdrawing substituents, suggesting that the enzyme stabilizes a partial positive charge at C-3. Binding to crotonase was shown to decrease the shielding of [3-13C,3-2H]cinnamoyl-CoA by +3.2 ppm, consistent with an increased partial positive charge at C-3. The Raman spectra of cinnamoyl-CoA bound at the crotonase active site similarly reflect the significant electronic ground state changes in the pi electronic structure of the bound substrate. These data show that a major rearrangement of electrons occurs in the acryloyl portion of the cinnamoyl group upon binding, while only a minor perturbation occurs to the distribution of electrons in the phenyl ring.(ABSTRACT TRUNCATED AT 250 WORDS)