Defining the Structure and Function of Enzymes

Histidine Triad Nucleotide Binding Proteins

Histidine Triad Binding Proteins (HINTs) are conserved from bacteria to human and are considered to be the ancestor of the histidine triad protein (HIT) superfamily. Humans express three HINTs: hHINT1, hHINT2 and hHINT3. Our laboratory has shown that hHINTs act as nucleoside phosphoramidate monoester and acyl-adenylate hydrolases and are therefore the likely i intracellular enzymatic activators of nucleoside phosphoramidates.

Ectopic expression studies and endogenous gene expression mapping have revealed that hHINT1 is typically found in the cytoplasm. hHINT1 has been found to be associated with transcription activation complexes, apoptosis regulation, neurotransmitter receptor modulation, mu-opioid receptor regulation, NMDAR regulation, cell differentiation and tumorigenicity and interactions with lysine tRNA synthetases. Substrate specificity analysis revealed that hHINT1 somewhat prefers phosphoramidates composed of purines over those composed of pyrimidines.

In addition, based on kinetic and crystallographic studies, the 2- and 3-hydroxyl groups of the substrate ribose ring were shown to be preferred for maximal phosphoramidase effi¬ciency. The most efficient phosphoramidate substrates were composed of the primary amine, tryptamine. The rapid turnover numbers (kcat) and low Km values exhibited by these substrates results in nearly diffusion-controlled apparent second-order rate constants (kcat/Km = 1.1-1.5 X 107 M-1 s-1). 

Recently, we have completed the first in depth characterization of the kinetic mechanism of hHINT1, demonstrating that adenylation of the enzyme is rapid and that both hydrolysis of the adenylated enzyme and product release are partially rate limiting. Given the active site sequence similarities between HINTs, the kinetic mechanism of hHINT1 should serve as a template for understanding catalysis by all HINT proteins.

While currently the role of HINT1 catalysis biological and it’s natural substrate, our group has demonstrated that bacterial HINTs regulate Alanine dehydrogenase, possibly regulate amino acid t-RNA transferases by efficiently hydrolyzing enzyme bound amino acid acyl-AMP and most interestingly regulate the interaction of the mu-opioid receptor with the NMDA receptor. This observation has lead us to develop inhibitors of HINT1 that are able to block opioid tolerance and neuropathic pain, identifying a new target for pain.

hHINT1 and hHINT2 have high (61%) sequence similarity. Like hHINT1, we have shown that human hHINT2 is a purine nucleotide phosphoramidase with kcat value of 6.7 s-1 and Km value of 4.03 uM, resulting in an apparent second-order rate constant (kcat/Km) for TpAd of 1.7 X 106. We have also demonstrated by size exclusion chromatography (SEC) and x-crystallography, in collaboration with Dr. Barry Finzel’s lab, that it is also a homodimer. hHINT2 is found exclusively in mitochondria and has been shown to be a mitochondrial apoptotic sensitizer, which is down-regulated in hepatocellular carcinomas. It also appears to be involved in the regulation of calcium-independent steroidogenesis. 

Little is known about HINT3’s. In contrast to hHINT1 and hHINT2, naturally or ectopically expressed hHINT3 is found in the cytoplasm, mitochondria and nucleus. Our laboratory was the first to purify hHINT3, revealing that hHINT3 is multimeric and not dimeric and prefers acyl-adenylated substrates over purine nucleotide phosphoramidates. We have concluded that hHINT3 is best classified as a distinct HIT sub-family and have identified a natural monomeric polymorph. 

Our laboratory is therefore actively seeking to determine the role of these ubiquitous enzymes in the plethora of biological functions they have been found to be engaged in by using the tools of chemical biology, molecular biology and pharmacology.

N-Acetytransferases (NATs)

Many chemicals that cause cancer or produce undesirable side effects do so only after they are converted (bioactivated) enzymatically to carcinogenic or toxic metabolites. Within this context, arylamine N-acetyltransferases (NATs) are versatile enzymes that catalyze both the conversion of carcinogenic arylamines, which are present ubiquitously in foods and chemical derivatives, to arylamides and the bioactivation of arylhydroxylamines and arylhydroxamic acids to electrophilic cancer causing metabolites.

Our laboratory, in collaboration with Dr. Patrick E. Hanna, cloned, expressed, and purified the hamster and human NAT isozymes and demonstrated a novel catalytic mechanism. We established the catalytic mechanism of this unique class of enzymes and elucidated the catalytic mechanism for this class of enzymes by steady-state and transient state kinetics, site-directed mutagenesis and active site labeling experiments. The results of our studies demonstrated that NAT-2 contains a highly reactive but solvent inaccessible catalytic quadrade of Cys-His-Asp-Tyr. Kinetic and solvent isotope effect studies clearly demonstrated that, although on the surface these enzymes resemble cysteine proteases, the catalytic mechanism utilizes the histidine and aspartate to facilitate proton deprotonation before the transition state for enzyme acetylation is reached. The stability of the acetylated enzyme is maximized by a combination of solvent sequestration and positioning of the catalytic histidine to assist in nucleophilic attack of arylamines, but not water or phenols. In contrast to cysteine proteases, site directed mutagenesis studies have indicated that these residues are intricately involved in both maintaining the catalytic and structural integrity of NAT’s.

In collaboration with Dr. Kylie Walters, we developed the first structure of a mammalian NAT with N-15 labeled protein and high field NMR. In addition, the ability of NAT’s to serve as a useful model system for the study of protein adducts was also extensively investigation. Chemical and molecular genetic and structural studies with hamster NAT’s and the recently cloned and purified human NAT-1 have allowed us to fully characterize the enzymatic role of these proteins in xenobiotic bioactivation, as well as, address the potential natural function of these enzymes. 

Key Accomplishments

HINTs:

• Elucidation of HINT1 catalytic and kinetic mechanism.

• Bacterial expression and purification of HINT2 and HINT3,

• X-ray structure of HINT2.

• Identified an unusual water channel in HINTs that regulates protein structure and enzymatic activity.

• Development of comprehensive substrate specificity analysis of HINT1 and HINT2.

• Design and development of cell permeable inhibitors of HINT1.

• Demonstration of that bacterial HINT regulates D-alanine metabolism by regulating D-alanine dehydrogenase.

• Demonstrated that HINT1 is able to hydrolyze amino acid T-RNA transferase bound amino acid acyl-AMP.

• Demonstrated that inhibitors of HINT1 are able to block as well as rescue mice from opioid tolerance and block neuropathic pain.

• Established a fluorescence on/off switch assay for HINTs.

• Elucidating the effect of natural mutations of HINT1 responsible for inherited peripheral neuropathy on enzyme structure and catalysis.

NATs:

• Established the first recombinant expression method for hamster and human NAT1 and NAT2 in collaboration with Dr. P.E. Hanna.

• Developed the first structural analysis of a mammalian NAT with high field NMR in collaboration with Dr. P.E. Hanna and Dr. Kylie Walters.

• Elucidated the catalytic and kinetic mechanism of NATs in collaboration with Dr. P.E. Hanna.

• Defined the mechanism of aryl amine nitroso ureas on NAT inactivation in collaboration with Dr. P.E. Hanna.

• Developed a substrate specificity profile for both NAT1 and NAT2 in collaboration with Dr. P.E. Hanna.