Matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Site (PICS) method to simultaneously profile both the prime and nonprime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with an ~32-93% preference for leucine in P1' depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and nonprime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. Thus these caveats make it critical to check for these highly biologically important cooperativity effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both a P3 proline and P1' leucine, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced nonprime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that nonprime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the nonprime and prime sides flanking the proteomic-pinpointed scissile bond.