Some have argued that catalytic antibody (abzyme) research challenges the argument for extreme protein rarity. In reality, the truth is the exact opposite. As a case study, Shahsavarian et al. used a phage display library of the size on the order of billions to generate catalytic antibodies approaching the efficiency of beta-lactamase enzymes in breaking down antibiotic molecules. Antibodies are highly specialized multicomponent proteins that are designed to maintain a stable structure as localized sections of the protein known as Fv regions dramatically vary. One Fv region resides at the end of each of the antibody’s two branches, and it consists of the variable domains within a heavy chain and a light chain. The immunoglobulin gene randomizes the variable regions allowing for a binding site to eventually appear that can bind to a target and possibly break it apart. Finding the right combination of amino acids to degrade an antibiotic molecule proved relatively easy.
Yet, abzymes function very differently from enzymes. In the former, the variable domains forming a binding site consist of localized sequences of amino acids held in fairly consistent positions by nonvarying sections known as constant regions. The constant regions also ensure the variable regions in the heavy and light chains reside at the right locations in close proximity. In contrast, an enzyme starts off with the amino acids which form the catalytic site residing at distant locations along the chain. The folding process forms the active site by moving the correct amino acids to the right locations and positioning them in the right orientations.
Moreover, in enzymes both the active site and amino acids throughout the protein structure are specified to assist in its target function. Specifically, an enzyme’s entire conformation morphs into multiple configurations. This complex dynamic is well summarized by Hammes et al.,
Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms.
Each reconfiguring involves the coordinated rearrangements of single amino acids and often entire secondary structures.
Therefore, the tasks of forming a functional abzyme and generating a novel functional enzyme represent fundamentally different problems. A new enzyme requires both finding a set of amino acids with the right chemical capacities and generating a new fold that brings those amino acids together properly in 3D space and provides structural support. The fold also must perform complex conformational changes to support specific chemical activities. The abzyme only needs to stumble upon the correct amino acid sequences in the variable regions for the catalytic activity. The amino acids are already positioned properly by the constant regions, and the latter also provide the needed structural support. In addition, abyzmes do not morph their overall conformations to assist specific chemical reactions. These differences explain abzymes’ limited capacities, and they result in enzymes having much greater functional sequence rarity.
Ironically, the abzyme research greatly strengthens the argument for the generality of extreme rarity, for it shows that degrading antibiotic molecules is a relatively easy function to achieve. In contrast, the enzyme HisA participates in an intermediate step in the synthesis of the amino acid histidine where it performs a highly specific molecular rearrangement. Namely, the enzyme detaches a hydrogen atom from one nitrogen molecule and attaches a hydrogen atom to another nitrogen. No abzyme or polypeptide generated in a randomized library has ever demonstrated a comparable ability to reengineer molecules.
The difference in the difficulty of antibiotic degradation and molecular reengineering explains beta-lactamase’s greater resilience to accumulating mutations than HisA’s. This difference directly translates into HisA’s more extreme sequence rarity. Many enzymes and structural proteins also perform more difficult tasks with greater specificity requirements than beta-lactamase, so a 10% populated target region should be an optimistic estimate for a large percentage of globular proteins.