PAICS Human

What is human PAICS and what is its functional significance?

Human PAICS is a bifunctional enzyme involved in de novo purine biosynthesis, catalyzing two consecutive reactions in the pathway. Specifically, PAICS converts aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR) through a two-step process . This enzyme comprises two distinct catalytic domains: AIR carboxylase (AIRc) and SAICAR synthetase (SAICARs), which work in concert to facilitate this transformation . The significance of PAICS extends beyond normal cellular metabolism, as it has been found to be overexpressed in various cancer types and is associated with enhanced cell proliferation, invasion, epithelial-mesenchymal transition, and efficient tumor growth . The crystal structure of human PAICS has been resolved at 2.8 Å resolution, providing crucial structural insights for understanding its catalytic mechanism .

How does human PAICS differ from PAICS in other organisms?

Human PAICS employs a unique catalytic strategy compared to PAICS in other organisms. In animals, including humans, PAICS contains a type II PurE domain (AIRc) that catalyzes the direct carboxylation of AIR to CAIR in a single step . This contrasts with the mechanism observed in plants, yeast, and most prokaryotes, which utilize a different pathway for CAIR synthesis . This evolutionary divergence highlights the specialized nature of human PAICS and has implications for the development of species-specific inhibitors. Additionally, experimental evidence suggests potential differences in substrate channeling mechanisms between human and avian PAICS, although the exact cause of this discrepancy remains unclear . These differences have significant implications for researchers designing experiments with model organisms when studying PAICS function.

What are the two catalytic activities of human PAICS and how do they function?

Human PAICS exhibits two distinct catalytic activities housed in separate domains:

• AIR carboxylase (AIRc) - This domain catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) to form carboxyaminoimidazole ribonucleotide (CAIR). The reaction involves the direct addition of CO₂ to AIR at the C4 position . Quantum chemical calculations have revealed that this reaction proceeds through a two-step mechanism: first, C-C bond formation occurs, followed by deprotonation of the formed tetrahedral intermediate (isoCAIR) assisted by an active site histidine residue .

• SAICAR synthetase (SAICARs) - This domain catalyzes the ATP-dependent condensation of CAIR with L-aspartate to form SAICAR. The reaction proceeds with the phosphorylation of CAIR occurring before the condensation reaction with aspartate . Three active site magnesium ions play crucial roles in binding the substrates and stabilizing the transition states and intermediates of the reaction .

These two catalytic activities work in tandem, with evidence suggesting that substrate channeling occurs between the domains, allowing efficient transfer of the CAIR intermediate from the AIRc to the SAICARs active site .

What evidence supports substrate channeling between domains of human PAICS?

Substrate channeling—the direct transfer of intermediates between enzyme active sites without release into bulk solution—has been demonstrated in human PAICS through multiple experimental approaches. Time-course mass spectrometric analysis provides compelling evidence for this mechanism. When CAIR is added to the bulk solution (CAIR bulk), it undergoes decarboxylation and re-carboxylation before SAICAR accumulation is observed . Experiments utilizing ¹³C-bicarbonate have shown that SAICAR production correlates directly with re-carboxylated CAIR rather than with total CAIR or CAIR bulk . This indicates that the SAICAR synthase domain preferentially utilizes enzyme-generated CAIR over CAIR from the bulk solution, consistent with a channeling model.

The channeling hypothesis is further supported by crystallographic structures of human PAICS, which reveal spatial arrangements conducive to intermediate transfer . Additionally, inhibition studies with NAIR (a nonreactive CAIR analogue) and mutation analyses of AIR carboxylase active site residues have demonstrated that altering the AIR carboxylase domain partially affects SAICAR synthase activity, reinforcing the interconnected nature of these domains .

This substrate channeling mechanism may represent part of a larger channeling process in de novo purine nucleotide synthesis, suggesting an efficient metabolic organization that minimizes intermediate diffusion and potential side reactions.

What are the detailed reaction mechanisms of human PAICS as elucidated by quantum chemical calculations?

Quantum chemical calculations have provided remarkable insights into the reaction mechanisms of human PAICS at an atomic level. For the AIR carboxylase reaction, density functional theory calculations have revealed a two-step mechanism:

• C-C Bond Formation: The nucleophilic C4 atom of AIR attacks the electrophilic carbon of CO₂, forming a tetrahedral intermediate known as isoCAIR .

• Deprotonation: An active site histidine residue (His303) assists in the deprotonation of the formed tetrahedral intermediate, completing the conversion to CAIR .

For the SAICAR synthetase reaction, the calculations demonstrate a sequential process:

• CAIR Phosphorylation: ATP phosphorylates CAIR, which occurs before the condensation reaction with aspartate .

• Nucleophilic Attack: The neutral amino group (-NH₂) of aspartate acts as a nucleophile in the reaction .

• Magnesium Ion Coordination: Three active site magnesium ions (designated as Mg A, Mg B, and Mg C) play critical roles in binding substrates and stabilizing transition states and intermediates . Their positions were identified through structural superposition with E. coli SAICARs complexed with ADP and CAIR .

The calculated energy barriers for these reactions align well with available experimental kinetic data, validating the computational approach and providing a comprehensive understanding of PAICS catalysis.

How can monoclonal antibodies against human PAICS be generated and utilized in cancer research?

The generation of monoclonal antibodies (mAbs) against human PAICS represents a valuable tool for both basic research and potential diagnostic applications. A recent study detailed the generation of rat mAb 6A10 specific for human PAICS . The development process involved:

The utility of such antibodies in cancer research extends to:

• Studying PAICS Expression: Enabling detection of PAICS overexpression in various cancer types.

• Functional Analyses: Facilitating investigations into the role of PAICS in cancer cell proliferation, invasion, and epithelial-mesenchymal transition.

• Diagnostic Applications: Potential use in diagnosing malignant transformation based on PAICS expression patterns .

These antibodies can help elucidate the mechanistic connection between PAICS overexpression and cancer progression, potentially leading to novel therapeutic strategies targeting this enzyme.

What crystallographic methods have been employed to determine human PAICS structure?

The crystal structure of human PAICS has been determined at 2.8 Å resolution, representing the first structure in the entire PAICS family . The crystallographic approaches used to elucidate this structure provided essential insights for rational anticancer drug design, as rapidly dividing cancer cells heavily rely on the purine de novo pathway for adenine and guanine synthesis .

More recent crystallographic studies have resolved human PAICS in complex with various ligands, including:

• PAICS-CAIR Complex: Human PAICS in complex with carboxyaminoimidazole ribonucleotide (CAIR) (PDB 6YB8) .

• PAICS-SAICAR-AMP-PNP Complex: Human PAICS in complex with SAICAR and AMP-PNP (an ATP analogue) (PDB 6YB9) .

These structures have allowed researchers to:

• Identify key residues involved in substrate binding and catalysis

• Understand the spatial relationship between the two active sites

• Develop models for substrate channeling between domains

• Provide templates for structure-based drug design efforts

The crystallographic models have been further enhanced through computational approaches, including the building of active site models for quantum chemical calculations. These models encompass the amino acids composing the active sites and interacting with substrates, providing a comprehensive view of the reaction environments .

How can mass spectrometry be utilized to study substrate channeling in human PAICS?

Mass spectrometry has proven instrumental in providing evidence for substrate channeling in human PAICS through innovative experimental designs:

• Time-Course Analysis: Mass spectrometric time-course analysis has been employed to monitor the fate of CAIR added to bulk solution versus enzyme-generated CAIR. This approach revealed that bulk CAIR undergoes decarboxylation and re-carboxylation before SAICAR accumulation, suggesting a selective mechanism .

• Isotope Labeling Studies: Experiments utilizing ¹³C-bicarbonate have demonstrated that SAICAR production correlates specifically with re-carboxylated CAIR rather than total CAIR or bulk CAIR. This selective incorporation of the labeled carbon provides strong evidence for the channeling model .

The experimental protocol typically involves:

This methodology allows researchers to distinguish between intermediates processed through the channeling pathway versus those from bulk solution, providing a powerful approach to study the kinetic advantages of substrate channeling in multi-domain enzymes .

What evidence supports PAICS as a target for cancer chemotherapy?

PAICS has emerged as a promising target for cancer chemotherapy based on several lines of evidence:

• Differential Expression: PAICS is overexpressed in various types of cancer, including acute myeloid leukemia, where it has been identified as a potential chemotherapy target through both in vitro and in vivo studies using mouse xenograft models .

• Metabolic Dependency: Rapidly dividing cancer cells rely heavily on the de novo purine biosynthesis pathway (which includes PAICS) for synthesis of adenine and guanine, whereas normal cells predominantly utilize the salvage pathway . This metabolic difference provides a potential therapeutic window.

• Functional Roles in Cancer: PAICS has been linked to multiple cancer-promoting processes, including:

  • Enhanced cell proliferation

  • Increased cellular invasion

  • Promotion of epithelial-mesenchymal transition (EMT)

  • Support of efficient tumor growth

• Structural Insights: The crystal structure of human PAICS provides essential information for designing specific inhibitors that could disrupt its function in cancer cells . The bifunctional nature of PAICS offers multiple sites for potential therapeutic intervention.

This accumulating evidence positions PAICS as an attractive target for developing novel anticancer agents that may exhibit selectivity for cancer cells over normal tissues, potentially minimizing off-target effects.

What methodological approaches can be used to develop specific inhibitors for human PAICS?

The development of specific inhibitors for human PAICS represents a promising avenue for cancer therapeutics. Several methodological approaches can be employed:

• Structure-Based Drug Design: Utilizing the crystal structures of human PAICS (such as PDB 2H31, 6YB8, and 6YB9) to identify potential binding pockets and design molecules that can specifically interact with catalytic residues . Virtual screening of compound libraries against these structures can identify lead candidates.

• Mechanism-Based Inhibitor Design: Developing transition state analogues or substrate mimics based on the elucidated reaction mechanisms from quantum chemical calculations . For example, designing stable analogues of the tetrahedral intermediate (isoCAIR) for the AIRc reaction.

• Exploitation of Substrate Channeling: Designing molecules that disrupt the substrate channeling between the AIRc and SAICARs domains, potentially offering a unique mechanism of inhibition not applicable to monofunctional enzymes .

• Targeted Antibody Development: Generating inhibitory monoclonal antibodies against specific epitopes of PAICS, building upon approaches used to develop research antibodies like mAb 6A10 .

• Rational Modification of Known Inhibitors: NAIR (a nonreactive CAIR analogue) has been shown to inhibit the AIR carboxylase activity of human PAICS . Structural modifications of this compound could enhance potency and specificity.

The experimental validation of potential inhibitors would typically involve:

• Enzymatic assays with recombinant human PAICS

• Cell-based assays in cancer cell lines with high PAICS expression

• Selectivity screening against related enzymes

• In vivo efficacy studies in appropriate cancer models

These approaches, guided by detailed structural and mechanistic understanding, could lead to the development of novel PAICS inhibitors as potential anticancer agents.

What are the current challenges and future opportunities in human PAICS research?

Despite significant advances in understanding human PAICS, several challenges and opportunities remain for future research:

• Resolving Mechanistic Discrepancies: Clarifying the discrepancies between studies of human and avian PAICS regarding substrate channeling and inhibitor specificity. While human PAICS shows evidence of channeling, earlier work with avian PAICS suggested otherwise . Resolving these species-specific differences would advance our understanding of evolutionary adaptations in purine biosynthesis.

• Improving Specificity of Inhibitors: Developing highly specific inhibitors that can distinguish between human PAICS and related enzymes to minimize off-target effects in potential therapeutic applications. The unique bifunctional nature of human PAICS presents both challenges and opportunities for selective targeting.

• Understanding Regulation Mechanisms: Elucidating how PAICS expression and activity are regulated in normal versus cancer cells. While overexpression has been documented in various cancers , the regulatory mechanisms controlling this upregulation remain poorly understood.

• Exploring the Extended Purinosome: Investigating how PAICS functions within the larger context of the purinosome—a multi-enzyme complex involved in purine biosynthesis. Evidence suggests that substrate channeling in PAICS may be part of a larger channeling process in de novo purine nucleotide synthesis .

• Translational Applications: Bridging the gap between basic PAICS research and clinical applications, particularly in cancer diagnostics and therapeutics. The development of monoclonal antibodies against PAICS represents one step in this direction , but further work is needed to realize the full potential of PAICS as a clinical target.

Future research employing integrated approaches combining structural biology, enzymology, cellular biology, and computational methods will be essential to address these challenges and capitalize on the opportunities presented by this important metabolic enzyme.